Members of TNF and TNFR families

ABSTRACT

New members of the TNF and the TNFR superfamily of proteins have been identified. These proteins are promising targets for therapeutic intervention and mimesis. TNF-L and TNFR-L proteins can be used to induce cell death and/or proliferation of cells. Members of these superfamilies have been implicated in a broad variety of disease processes, making them central biological and physiological regulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of application Ser. No.60/068,959 filed Dec. 30, 1997, and of application Ser. No. 09/212,270,filed Dec. 16, 1998, both of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Tumor necrosis factor (TNF) is a pro-inflammatory cytokine whichis produced by a wide spectrum of cells. It has a key role in hostdefense and immunosurveillance, mediating complex cellular responses. Inexcess, TNF may have detrimental effects.

[0003] Two specific, high affinity cell surface receptors, p55 TNF-R andp75 TNF-R, function as transducing elements, providing the intracellularsignal for cell responses to TNF. While both types of TNF receptors areexpressed by almost all cell types, the p75 receptor has been shown tobe expressed primarily by cells of the immune system (B and T cells),cells of myeloid origin, and endothelial cells. Both receptorsparticipate in the induction of NFκB and interleukin-6, in thegeneration of lymphocyte activated killer (LAK) cells, and in theproliferation of natural killer (NK) cells, as well as inanti-proliferation, cytotoxicity, and apoptosis.

[0004] TNF signaling to cells is largely mediated by the p55 TNF-R,while the main function of the p75 surface receptor is “ligand passing,”i.e., TNF presentation to the p55 TNF-R. Presence of the cell surfacep75 TNF receptor greatly enhances the rate of association of TNF to thep55 TNF receptor and may reverse the desensitization of p55 TNF-R toTNF. Pharmaceutical agents which affect p75 TNF-R may have a generalimpact on TNF function, including those activities in which the majorsignaling receptor is the p55 TNF-R.

[0005] The TNF-Rs also mediate many non-overlapping functions: the p55receptor is involved in interleukin-2 receptor induction, anti-viralactivities, growth stimulation, HLA antigen expression, and endothelialcell adhesion, while the p75 receptor mediates the TNF-induced thymocyteproliferation.

[0006] The p55 and p75 TNF-Rs are members of a superfamily whichincludes nerve growth factor receptor (NGFR), Fas antigen, CD27, CD30,CD40, OX40 and 4-1BB. The cysteine-rich domains of the extracellularpart of these receptors are homologous to several viral proteinsproduced by cowpox virus, Shope fibroma virus, and the myxoma virus.

[0007] Because of the central role of TNF and its receptors in hostdefense and immunosurveillance, there is a need in the art to identifynew members of the TNF and TNFR superfamilies

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide new members of theTNF and TNFR families, as well as methods of screening for compoundscapable of modifying the activities of these proteins. This and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

[0009] One embodiment of the invention is an isolated human proteinhaving an amino acid sequence which is at least 85% identical to anamino acid sequence selected from the group consisting of SEQ ID NOS:1,2, 17 and 20. Percent identity is determined using a Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 1.

[0010] Another embodiment of the invention is a fusion proteincomprising a first protein segment and a second protein segment fusedtogether by means of a peptide bond. The first protein segment consistsof a protein having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 2, 17 and 20.

[0011] Still another embodiment of the invention is a preparation ofantibodies which specifically bind to a protein having an amino acidsequence selected from the group consisting of SEQ ID NOS:1, 2, 17 and20.

[0012] Even another embodiment of the invention is a cDNA molecule whichencodes a protein having an amino acid sequence which is at least 85%identical to an amino acid sequence selected from the group consistingof SEQ ID NOS:1, 2, 17 and 20. Percent identity is determined using aSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 1.

[0013] Yet another embodiment of the invention is a cDNA molecule whichis at least 85% identical to a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:6, 7, 18 and 19. Percent identity isdetermined using a Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 1.

[0014] A further embodiment of the invention is an isolated and purifiedsubgenomic polynucleotide comprising a nucleotide sequence whichhybridizes to a nucleotide sequence selected from the group consistingof SEQ ID NOS:6, 7, 18 and 19 after washing with 0.2×SSC at 65° C. Thenucleotide sequence encodes a protein having an amino acid sequenceselected from the group consisting of SEQ ID NOS:1, 2, 17and 20.

[0015] Another embodiment of the invention is a construct comprising apromoter and

[0016] a polynucleotide segment encoding an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:1, 2, 17 and 20 Thepolynucleotide segment is located downstream from the promoter.Transcription of the polynucleotide segment initiates at the promoter.

[0017] Still another embodiment of the invention is a host cellcomprising a construct which comprises a promoter and a polynucleotidesegment encoding an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 2, 17 and 20.

[0018] Even another embodiment of the invention is a recombinant hostcell comprising a new transcription initiation unit, wherein the newtranscription initiation unit comprises in 5′ to 3′ order (a) anexogenous regulatory sequence, (b) an exogenous exon, and (c) a splicedonor site. The new transcription initiation unit is located upstream ofa coding sequence of a gene. The coding sequence is selected from thegroup consisting of SEQ ID NOS:6, 7, 18 and 19. The exogenous regulatorysequence controls transcription of the coding sequence of the gene.

[0019] Yet another embodiment of the invention is a method of screeningfor a compound capable of modulating cell death inducing activity of aprotein. A first population of cells and a protein are incubated in thepresence of a test compound. The protein comprises an amino acidsequence selected from the group of amino acid sequences shown in SEQ IDNOS:1-5, 17 and 20. A second population of cells and the protein areincubated in the absence of a test compound. Viability of the first andsecond populations is determined. A test compound which increases ordecreases viability of the first population relative to the secondpopulation is identified as capable of modulating the cell deathinducing activity of the protein.

[0020] Even another embodiment of the invention is a method ofidentifying a binding partner of a first protein. A first protein isincubated with a second protein. The first protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS:1-5, 17and 20 Formation of a complex between the first and second proteins isdetected. Formation of the complex identifies the second protein as abinding partner of the first protein.

[0021] The present invention thus provides the art with the amino acidsequences of proteins which are new members of the TNF and TNFRfamilies, as well as nucleotide sequences of polynucleotides whichencode the proteins. These proteins and polynucleotides can be used toenhance or decrease TNF activities thereby providing therapeuticbenefits, such as induction of cell death, lymphoid organogenesis, orhost bacterial resistance, and inhibition of endotoxic shock, contacthypersensitivity, delayed type hypersensitivity, or immunocompetence ofa transplant recipient. Methods of diagnosing neoplasia orpredisposition to neoplasia are also provided. Proteins of the presentinvention are also useful for identifying compounds which can regulatethe TNF-like or TNFR-like activities of these proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1. FIG. 1A shows the protein sequence of human TNFL1. Thetransmembrane domain is underlined. FIG. 1B shows alignments of theextracellular domains of several members of the TNF family with TNFL1.The conserved amino acids are boxed.

[0023]FIG. 2. FIG. 2A shows TNFL1 mRNA expression in human tissues andcell lines. FIG. 2B shows TNFL1 expression in mouse tissues PBL,peripheral blood lymphocytes; HL60 promyelocytic leukemia HL60; HeLa,HeLa cell line S3; K562, chromic myelogenous leukemia K562; MOLT-4,lymphoblastic leukemia MOLT-4; Raji, Burkitt's lymphoma Raji; SW480,colorectal adenocarcinoma SW480; A549, lung carcinoma A549; G361,melanoma G361.

[0024]FIG. 3. FIG. 3 shows detection of TNFL1 expression using apurified polyclonal anti-peptide antibody.

[0025]FIG. 3A shows a Western blot analysis using the affinity-purifiedD2710 antibody at a final concentration of 1 μg/ml. The extractsanalyzed were total cell extracts of human monocytes isolated from PBMCs(lanes 1 and 2) and mouse bone marrow-derived dendritic cells (lanes 3and 4). Lanes 5 and 6 contained 20 ng of TNFL1 protein purified from E.coli In lanes 2, 4, and 6 the antibody was pre-incubated with ahundred-fold molar excess of the peptide used to generate the antibody.

[0026]FIG. 3B shows intracellular staining of baculovirus-infected cellsexpressing TNFL1, using the D2710 antibody or a control rabbit antibodyat a concentration of 10 μg/ml. The insect cells were fixed andpermeabilized before staining and flow cytometry analysis.

[0027]FIG. 3C shows the pattern of expression of TNFL1 in mouse spleensections. Left panels: rabbit antibody control plus secondary antibody;right panels, anti-TNFL1 antibody plus secondary antibody. Themagnification is 100x in the top panels and 200x in the bottom panels.RP, red pulp; PALS, periarteriolar lymph sheath.

[0028]FIG. 3D shows staining of adjacent murine spleen sections forThy-1.2, B220, TNFL1, CD11c, and Mac-3.

[0029]FIG. 4. FIG. 4 shows flow cytometric analysis of the cell-surfaceexpression of TNFL1 on sub-populations of human PBMCs and culturedcells.

[0030]FIG. 4A shows expression of TNFL1 on CD14+, CD19+, CD4+, and CD8+human PBMCs and on mouse bone marrow-derived dendritic cells. HumanPBMCs were stained with affinity purified D2710, followed byFITC-conjugated anti-rabbit IgG. They were subsequently stained withPE-conjugated CD14, CD19, CD4, or CD8. The histograms show TNFL1expression (FITC) on PE-positive gated cells. The dotted lines representthe staining in the absence of primary antibody D2710.

[0031]FIG. 4B shows upregulation of TNFL1 surface expression on T cellsafter activation with anti-CD3 and anti-CD28 at 10 μg/ml in the presenceof IL2 at 50 μg/ml for six days.

[0032]FIG. 5. FIG. 5 shows the effect of recombinant TNFL1 on activatedT and B cells.

[0033]FIG. 5A shows the amino acid sequence of a soluble form of TNFL1(sequence enclosed in brackets) which was expressed in E. coli as afusion protein and used for all biological assays. The cleavage sitesidentified after microsequencing of the truncated form of the proteinare represented as vertical bars.

[0034]FIG. 5B shows inhibition of DNA synthesis in activated but not inresting T cells by TNFL1. T cells enriched from human PBMCs were eitheractivated with 10 μg/ml of anti-CD3 and 10 μg/ml of anti-CD28 (leftpanel) or untreated (right panel) for 24 hours. TNFL1 was then added atvarious concentrations, and ³H-thymidine incorporation over a period of8 hours was measured 48 hours after addition of TNFL1.

[0035]FIG. 5C shows inhibition of DNA synthesis by TNFL1 in activated Bcells and activated T cells. B cells were activated with 10 μg/ml ofanti-CD40 antibody. T cells were activated as described in FIG. 5B.TNFL1 was added 48 hours after activation. ³H-thymidine incorporationover a period of 8 hours was measured 48 hours after addition of TNFL1.

[0036]FIG. 5D shows that TNFL1 induces apoptosis of activated T cells. Tcells were activated with anti-CD3 and anti-CD28 for 48 hours. TNFL1 wasadded at 2 μg/ml and incubated for an additional 48 hours. Apoptosis wasassessed by TUNEL assay. (R1), blasting cells; (R2), apoptotic cells;(R3), resting non-apoptotic cells.

[0037]FIG. 5E shows that TNFL1 induces NFκB activation in Jurkat cells.An electrophoretic mobility shift assay was performed with an NFκB probeon nuclear cell extracts prepared from Jurkat cells treated with PMA (1μg/ml) or TNFL1 (3 μg/ml) for one hour. Wild-type (wt, 20 ng) andmutated (mut, 100 ng) non-radiolabeled oligonucleotides were used ascompetitors in the reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0038] New members of the TNF and TNFR families are a discovery of thepresent invention. In particular, the present invention identifies twocDNA clones which encode new members of TNF ligand family and three cDNAclones which encode new members of TNFR family. Proteins andpolynucleotides of the invention provide the art with diagnostic andtherapeutic reagents as well as tools for discovering other therapeuticagents.

[0039] Two cDNA clones have been identified which show significanthomology to members of TNF family. One cDNA clone (SEQ ID NO:6) wasisolated from a human liver cDNA library. It encodes a proteindesignated as TNFL1 (SEQ ID NO:1), which has 27%-30% homology with TNFand 38% homology with lymphotoxin. TNFL1 contains a transmembranedomain. The TNFL1 cDNA clone encodes a 3 kb mRNA which can be detectedin tissues associated with the immune system, such as peripheral bloodlymphocytes, spleen, and thymus, as well as small intestine and ovary.TNFL1 protein is constitutively expressed on monocytes and B cellsisolated from human peripheral blood lymphocytes, as well as on mousedendritic cells and in mouse spleen. Expression of the protein can beup-regulated in natural killer cells. Activation of dendritic cells, forexample with anti-CD4d antibody, can down-regulate expression of TNFL1protein.

[0040] TNFL1 shares some common features with other members of the TNFfamily TNFL1 is upregulated on activated T cells, as are TNF, Fasligand, and CD30 ligand TNFL1 induces activation of NFκB, which is alsotriggered by every member of the TNF-R family with the exception of Fasand DR4. TNFL1 also leads to apoptosis of activated T cells, a welldocumented effect in the case of Fas ligand and TNF. TNFL1 differs inits expression pattern, however, when compared to TNF or Fas ligand. Fasligand, which is involved in activation-induced cell death (AICD) ofCD4+ T cells and tolerance to self-antigens, is classically expressed onactivated T cells and the immuno-privileged eye and testis. TNF isconstitutively expressed on both mature and immature thymocytes, isupregulated on activated T cells, and is induced by LPS on macrophages.In contrast, TNFL1 is constitutively expressed on antigen presentingcells, specialized or not, such as monocytes, B cells from peripheralblood lymphocytes in humans, and likely on macrophages or dendriticcells in the red pulp and marginal zone of mouse spleen and cultureddendritic cells.

[0041] This localization in the blood and in the spleen suggests apossible function for TNFL1 in the recognition process of blood-bornepathogens, such as bacteria or viruses. Furthermore, TNFL1 is expressedat the surface of dendritic cells cultured from bone marrow in thepresence of GM-CSF TNFL1 may therefore be expressed on a subtype ofdendritic cells which were recently individualized, myeloid dendriticcells (MDCs), rather than on lymphoid dendritic cells (LDCs). MDCs sharea precursor with macrophages, are GM-CSF dependant, and are present inthe marginal zone of secondary lymphoid tissues. LDCs, in contrast, arelocated in the T-cell zone of the secondary organs, are IL3-dependent,and share a precursor with T and B cells.

[0042] A second cDNA clone was isolated from an oligodT-primed libraryof a human ovarian tumor. It encodes a protein designated as TNFL2,which has about 25% homology with TNF. TNFL2 does not have atransmembrane domain and thus can be secreted. The sequence of TNFL2 isshown in SEQ ID NO:5 The TNFL2 cDNA clone detects a major population ofmRNA in a range of about 1.5 kb to about 2 kb in tissues associated withthe immune system, e.g., peripheral blood lymphocytes and spleen. Aslightly bigger mRNA is also expressed in spleen as well as in colon,prostate, and to a lesser extent in ovary and small intestine. The filllength polynucleotide sequence of TNFL2 cDNA is shown in SEQ ID NO:10

[0043] TNFL1 might be able to bind to the TNF receptors or to Fas, as ahomotrimer or in association with another member of the TNF family.TNFL1 and TNFL2 may form heterodimers and work together in a mannersimilar to that of lymphotoxin α and β. The TNF-like ligands disclosedherein can be used, inter alia, to induce cell death in tumors, toinduce apoptosis of activated T cells, to induce inflammation, and torescue resting T cells from apoptosis.

[0044] Proteins which are members of the TNFR superfamily have also beenidentified. These are soluble receptors which have the amino acidsequences shown in SEQ ID NOS:2 and 3 (human) and SEQ ID NO:4 (mouse).These proteins are encoded by the nucleotide sequences shown in SEQ IDNOS:7, 8, and 9, respectively. These receptors can be used, inter alia,to regulate the function of a TNF ligand which plays a role inapoptosis, inflammation, differentiation, or proliferation. Expressionof the receptors can also be useful as markers for cancer, especiallyfor colon cancer. Diseases which can be treated using the ligands and/orreceptors of the TNF/TNFR superfamily include rheumatoid arthritis,cancer, septic shock, Crohn's disease, and osteoporosis.

[0045] Two forms of the soluble receptor corresponding to SEQ ID NO:2have been identified. The first (tnfrGT-1) has 300 amino acids, as shownin SEQ ID NO:17. The second form (tnfrGT-2) is shown in SEQ ID NO:20.Polynucleotides encoding these two forms are shown SEQ ID NOS:18 and 19,respectively.

[0046] The human TNF-like (TNF-L) and mammalian TNF receptor-like(TNFR-L) proteins or polypeptides, biologically active polypeptides orprotein variants, and fusion proteins disclosed herein can be used invarious therapeutic compositions and methods, as described below. Anynaturally occurring variants of SEQ ID NOS:1-5 which may occur in humanor mammalian tissues and which retain the functional properties of theTNF-L or TNFR-L proteins disclosed herein are biologically active TNF-Lor TNFR-L variants. Non-naturally occurring TNF-L or TNFR-L variantswhich contain conservative amino acid substitutions relative to SEQ IDNOS:1-5 but which retain substantially the same ligand or receptoractivity as naturally occurring TNF-L or TNFR-L are also biologicallyactive TNF-L or TNFR-L variants.

[0047] Naturally or non-naturally occurring TNF-L or TNFR-L variantspreferably are at least 85%, 90%, or 95% identical to SEQ ID NOS 1-5 andhave similar biological functions, which are described below. Morepreferably, the molecules are 98% or 99% identical. Percent identity isdetermined using the Smith-Waterman homology search algorithm, using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 1. The Smith-Waterman homology search algorithm is taught inSmith and Waterman, Adv. Appl. Math. (1981) 2:482-489.

[0048] Biologically active TNF-L or TNFR-L variants include glycosylatedforms of the proteins, aggregative conjugates of the proteins with othermolecules, and covalent conjugates of the proteins with unrelatedchemical moieties. Covalent conjugates are prepared by linkage offunctionalities to groups which are found in the amino acid chain or atthe N- or C-terminal residues of the proteins of the invention by meanswell known in the art. TNF-L or TNFR-L variants also include allelicvariants, species variants, and muteins. Truncations or deletions ofregions which do not affect the biological functions of the TNF-L orTNFR-L proteins disclosed herein are also biologically active TNF-L orTNFR-L variants.

[0049] A subset of mutants, called muteins, is a group of polypeptideswith the non-disulfide bond participating cysteines substituted with aneutral amino acid, generally, with serines. These mutants may be stableover a broader temperature range than naturally occurring TNF-L orTNFR-L proteins. See Mark et al., U.S. Pat. No. 4,959,314.

[0050] Biologically active TNF-L or TNFR-L polypeptides can comprise atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, or 275 contiguous amino acids of SEQ ID NO:1, atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, or150 contiguous amino acids of SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12,15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175, or 200 contiguousamino acids of SEQ ID NO:3, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 50, 75, 100, 125, 130, or 140 contiguous amino acids of SEQ IDNO:4, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75,100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO:5, atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, 200, 225, 230, 231, 240, 250, 275, or 295 contiguous aminoacids of SEQ ID NO:17, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or 210 contiguous aminoacids of SED ID NO:20. Polypeptide molecules having substantially thesame amino acid sequences as the TNF-L or TNFR-L proteins disclosedherein but possessing minor amino acid substitutions which do notsubstantially affect the ability of the TNF-L or TNFR-L polypeptides tointeract with their respective receptors or ligands are within thedefinition of biologically active TNF-L or TNFR-L polypeptide variants.

[0051] Preferably, biologically active TNF-L or TNFR-L polypeptides orpolypeptide variants are at least 65%, 75%, 85%, 90%, 95%, 98%, or 99%identical to TNF-L or TNFR-L polypeptide fragments of SEQ ID NOS:1-5, 17or 20. Percent identity of potential polypeptides or polypeptidevariants with fragments of SEQ ID NOS:1-5, 17 or 20 is determined asdescribed above.

[0052] Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, such as DNASTAR software. Preferably the amino acid changesin TNF-L or TNFR-L protein or polypeptide variants are conservativeamino acid changes, i.e., changes of similarly charged or unchargedamino acids. Conservative replacements are those which take place withina family of amino acids which are related in their side chains.Genetically encoded amino acids are generally divided into four familiesacidic (aspartate, glutamate); basic (lysine, arginine, histidine);non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and uncharged polar (glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids.

[0053] It is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding properties of the resulting TNF-L or TNFR-L molecule, especiallyif the replacement does not involve an amino acid at a binding siteinvolved in an interaction of a TNF-L protein with its receptor or aTNFR-L protein with its ligand. Binding between a TNF-L protein and itsreceptor or a TNFR-L protein and its ligand can be measured, forexample, using a yeast two-hybrid assay, as is known in the art (Fields& Song, Nature 340:245-46, 1989).

[0054] Alternatively, the amino acid sequence of a TNF-L or TNFR-Lprotein can be modified to alter its biological activity. For example,amino acids 174-193 (the TNF ligand binding domain) can be deleted in aTNFR-L protein to form an inactive variant of the TNFR-L protein andthereby inhibit or decrease the function of its ligand.

[0055] TNF-L or TNFR-L proteins or polypeptides can be isolated fromTNF-L and TNFR-L-producing cells, such as spleen, thymus, prostate,colon, ovary, small intestine, peripheral blood lymphocytes, or fromcell lines such as K562 (chronic myeoleukemia), G361 (melanoma), orSW480 (colorectal adenocarcinoma), using biochemical methods which arestandard in the art. These methods include, but are not limited to, sizeexclusion chromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, crystallization,electrofocusing, and preparative gel electrophoresis. The skilledartisan can readily select methods which will result in a preparation ofTNF-L or TNFR-L protein which is substantially free from other proteinsand from carbohydrates, lipids, or subcellular organelles. A preparationof isolated and purified TNF-L or TNFR-L protein is at least 80% pure;preferably, the preparations are 90%, 95%, or 99% pure Purity of thepreparations can be assessed by any means known in the art, such asSDS-polyacrylamide gel electrophoresis.

[0056] Human TNF-L and human or mammalian TNFR-L proteins, polypeptides,or variants can be produced by recombinant DNA methods or by syntheticchemical methods. For production of recombinant TNF-L or TNFR-L proteinsor polypeptides, coding sequences selected from the nucleotide sequencesshown in SEQ ID NOS:6-10, 18 and 19 can be expressed in knownprokaryotic or eukaryotic expression systems. Bacterial, yeast, insect,or mammalian expression systems can be used, as is known in the art.

[0057] Alternatively, synthetic chemical methods, such as solid phasepeptide synthesis, can be used to synthesize human TNF-L or human ormammalian TNFR-L protein, polypeptides, or variants. General means forthe production of peptides, analogs or derivatives are outlined inCHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS—ASURVEY OF RECENT DEVELOPMENTS, B. Weinstein, ed. (1983). Substitution ofD-amino acids for the normal L-stereoisomer of a TNF-L or TNFR-L proteinof the invention can be carried out to increase the half-life of themolecule.

[0058] Fusion proteins comprising at least 6, 7, 8, 9, 10, 12, 15, 20,25, 30, 35, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275contiguous amino acids of SEQ ID NO:1, at least 6, 7, 8, 9, 10, 12, 15,20, 25, 30, 35, 40, 50, 75, 100, 125, or 150 contiguous amino acids ofSEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50,75, 100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO:3, atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 130,or 140 contiguous amino acids of SEQ ID NO:4, at least 6, 7, 8, 9, 10,12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175, or 200contiguous amino acids of SEQ ID NO:5, at least 6, 7, 8, 9, 10, 12, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 230, 231,240, 250, 275, or 295 contiguous amino acids of SEQ ID NO:17, or atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, 200, or 210 contiguous amino acids of SEQ ID NO:20 can also beconstructed. TNF-L and TNFR-L fusion proteins are useful for generatingantibodies against TNF-L and TNFR-L amino acid sequences and for use invarious assay systems For example, TNF-L and TNFR-L fusion proteins canbe used to identify proteins which interact with these proteins whichinfluence their biological activity and/or ability to bind to theirrespective binding partners. Physical methods, such as protein affinitychromatography, or library-based assays for protein-protein interactionssuch as the yeast two-hybrid or phage display systems, can also be usedfor this purpose. Such methods are well known in the art and can also beused as drug screens.

[0059] A TNF-L or TNFR-L fusion protein comprises two protein segmentsfused together by means of a peptide bond. The first protein segmentconsists of at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, or 275 contiguous amino acids of SEQID NO:1, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75,100, 125, or 150 contiguous amino acids of SEQ ID NO:2, at least 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175, or 200contiguous amino acids of SEQ ID NO:3, at least 6, 7, 8, 9, 10, 12, 15,20, 25, 30, 35, 40, 50, 75, 100, 125, 130, or 140 contiguous amino acidsof SEQ ID NO:4, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50,75, 100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO:5, atleast 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, 200, 225, 230, 231, 240, 250, 275, or 295 contiguous aminoacids of SEQ ID NO:17, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or 210 contiguous aminoacids of SEQ ID NO:20 The amino acids can be selected from the aminoacid sequences shown in SEQ ID NOS:1-5, 17 or 20 or from a biologicallyactive variants of those sequences The first protein segment can also bea full-length TNF-L or TNFR-L protein comprising an amino acid sequenceas shown in SEQ ID NOS:1-5, 17 or 20. The first protein segment can beN-terminal or C-terminal, as is convenient.

[0060] The second protein segment can be a full-length protein or aprotein fragment or polypeptide. Proteins commonly used in fusionprotein construction include β-galactosidase, β-glucuronidase, greenfluorescent protein (GFP), autofluorescent proteins, including bluefluorescent protein (BFP), glutathione-S-transferase (GST), luciferase,horseradish peroxidase (HRP), and chloramphenicol acetyltransferase(CAT). Epitope tags can be used in fusion protein constructions,including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusionconstructions can include maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP 16 protein fusions.

[0061] According to one particularly preferred embodiment, a TNFR-Lprotein is fused to the Fc domain of an IgG1 molecule. Such a fusionprotein is be useful for inhibiting the action of a TNF ligand.

[0062] TNF-L or TNFR-L fusion proteins can be made by covalently linkingthe first and second protein segments or by standard procedures in theart of molecular biology. Recombinant DNA methods can be used to preparethe fusion proteins, for example, by making a DNA construct whichcomprises coding sequences selected from SEQ ID NOS:6-10, 18, and 19 inproper reading frame with nucleotides encoding the second proteinsegment and expressing the DNA construct in a host cell, as is known inthe art. Many kits for constructing fusion proteins are available fromcompanies which supply research labs with tools for experiments,including, for example, Promega Corporation (Madison, Wis.), Stratagene(La Jolla, Calif.), Clontech (Mountain View, Calif.), Santa CruzBiotechnology (Santa Cruz, Calif.), NML International Corporation (MIC;Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada,1-888-DNA-KITS).

[0063] Isolated TNF-L or TNFR-L proteins, polypeptides, biologicallyactive variants, or fusion proteins can be used as immunogens, to obtaina preparation of antibodies which specifically bind to epitopes of TNF-Lor TNFR-L proteins. The antibodies can be used, inter alia, to detectTNF-L or TNFR-L proteins in tissue of humans or other mammals or infractions thereof The antibodies can also be used to detect the presenceof mutations in genes which result in under- or over-expression of TNF-Lor TNFR-L proteins or in expression of a TNF-L or TNFR-L protein withaltered size or electrophoretic mobility By binding to TNF-L or TNFR-Lproteins, antibodies can also alter the binding properties or biologicalfunctions of the proteins, for example for therapeutic use.

[0064] Antibodies which specifically bind to epitopes of TNF-L or TNFR-Lproteins, polypeptides, fusion proteins, or biologically active variantscan be used in immunochemical assays, including but not limited toWestern blots, ELISAs, radioimmunoassay, immunohistochemical assays,immunoprecipitations, or other immunochemical assays known in the art.Typically, antibodies provide a detection signal at least 5-, 10-, or20-fold higher than a detection signal provided with other proteins whenused in such immunochemical assays. Preferably, antibodies whichspecifically bind to TNF-L or TNFR-L protein epitopes do not detectother proteins in immunochemical assays and can immunoprecipitate TNF-Lor TNFR-L proteins or polypeptides from solution.

[0065] TNF-L- or TNFR-L-specific antibodies specifically bind toepitopes present in a protein having the amino acid sequence shown inSEQ ID NOS:1-5, 17 and 20 or to biologically active variants of thosesequences. Typically, at least 6, 8, 10, or 12 contiguous amino acidsare required to form an epitope. However, epitopes which involvenon-contiguous amino acids may require more, e.g., at least 15, 25, or50 amino acids. Preferably, TNF-L or TNFR-L epitopes are not present inother proteins. A preferred epitope comprises amino acids 208-211 of SEQID NO:20. Antibodies capable of specifically binding to a proteincomprising this epitope are useful for identifying a protein expressedby the polynucleotide of SEQ ID NO:19.

[0066] Protein epitopes which are particularly antigenic can beselected, for example, by routine screening of polypeptides forantigenicity or by applying a theoretical method for selecting antigenicregions of a protein to the amino acid sequences shown in SEQ IDNOS:1-5. Such methods are taught, for example, in Hopp and Wood, Proc.Nat. Acad. Sci. U.S.A. 78, 3824-28 (1981), Hopp and Wood, Mol. Immunol.20, 483-89 (1983), and Sutcliffe et al., Science 219, 660-66 (1983).

[0067] Any type of antibody known in the art can be generated to bindspecifically to TNF-L or TNFR-L epitopes. For example, preparations ofpolyclonal and monoclonal antibodies can be made using standard methodswhich are well known in the art Similarly, single-chain antibodies canalso be prepared. Single-chain antibodies which specifically bind toTNF-L or TNFR-L epitopes can be isolated, for example, from asingle-chain immunoglobulin display library, as is known in the art. Thelibrary is “panned” against the amino acid sequences disclosed herein,and a number of single chain antibodies which bind with high-affinity todifferent epitopes of proteins of the invention can be isolated. Hayashiet al., 1995, Gene 160:129-30. Single-chain antibodies can also beconstructed using a DNA amplification method, such as the polymerasechain reaction (PCR), using hybridoma cDNA as a template. Thirion etal., 1996, Eur. J. Cancer Prev. 5.507-11.

[0068] Single-chain antibodies can be mono- or bispecific, and can bebivalent or tetravalent. Construction of tetravalent, bispecificsingle-chain antibodies is taught, for example, in Coloma and Morrison,1997, Nat. Biotechnol. 15: 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught inter alia in Mallender and Voss,1994, J. Biol. Chem. 269:199-206.

[0069] A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology. Verhaar et al., 1995,Int. J. Cancer 61:497-501, Nicholls et al., 1993, J. Immunol. Meth.165:81-91.

[0070] Monoclonal and other antibodies can also be “humanized” in orderto prevent a patient from mounting an immune response against theantibody when it is used therapeutically. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or may require alteration of a few key residues. Sequencedifferences between, for example, rodent antibodies and human sequencescan be minimized by replacing residues which differ from those in thehuman sequences, for example, by site directed mutagenesis of individualresidues, or by grafting of entire complementarity determining regionsAlternatively, one can produce humanized antibodies using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto TNF-L or TNFR-L epitopes can contain antigen binding sites which areeither partially or fully humanized, as disclosed in U.S. Pat. No.5,565,332.

[0071] Other types of antibodies can be constructed and used in methodsof the invention. For example, chimeric antibodies can be constructed asdisclosed, for example, in WO 93/03151. Binding proteins which arederived from immunoglobulins and which are multivalent andmultispecific, such as the “diabodies” described in WO 94/13804, canalso be prepared

[0072] Antibodies can be purified by methods well known in the art. Forexample, antibodies can be affinity purified by passing the antibodiesover a column to which a TNF-L or TNFR-L protein, polypeptide,biologically active variant, or fusion protein is bound. The boundantibodies can then be eluted from the column, using a buffer with ahigh salt concentration.

[0073] TNF-L- or TNFR-L-specific binding polypeptides other thanantibodies can also be identified. These polypeptides include ligands ofTNFR-L proteins and receptors of TNF-L proteins. TNF-L- orTNFR-L-specific binding polypeptides are polypeptides which bind withTNF-L or TNFR-L proteins or their variants and which have a measurablyhigher binding affinity for TNF-L or TNFR-L and polypeptide variants ofthese proteins than for other polypeptides tested for binding. Higheraffinity by a factor of 10 is preferred, more preferably a factor of100. Such polypeptides can be found, for example, using the yeasttwo-hybrid system.

[0074] Nucleotide sequences which encode TNF-L or TNFR-L proteins areshown in SEQ ID NOS:6-10, 18 and 19 Isolated and purifiedpolynucleotides according to the invention can be single- ordouble-stranded, are subgenomic, and contain less than a wholechromosome. Preferably, the subgenomic polynucleotides are intron-free.

[0075] Isolated and purified subgenomic polynucleotides according to theinvention can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, or 1200 contiguousnucleotides of SEQ ID NO:6, at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, or 450 contiguous nucleotides of SEQ ID NO:7, at least10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575,600, 650, 700, 750, 800, or 850 contiguous nucleotides of SEQ ID NO:8,at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 575, or 600 contiguous nucleotides of SEQ ID NO:9, or at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 1100, or 1200 contiguous nucleotides of SEQ ID NO:10, atleast 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600,1700, 1800, or 1830 contiguous nucleotides of SEQ ID NO:19, or cancomprise SEQ ID NOS:6, 7, 8, 9, 10, 18, or 19. Such polynucleotides canbe used, for example, as primers or probes or for expression of TNF-L orTNFR-L proteins or polypeptides.

[0076] The complements of the nucleotide sequences shown in SEQ IDNOS:6-10, 18, and 19 are contiguous nucleotide sequences which formWatson-Crick base pairs with a contiguous nucleotide sequence as shownin SEQ ID NOS:6-10, 18, and 19. The complements of SEQ ID NOS:6-10, 18,and 19 are polynucleotides of the invention and can be used, forexample, to provide antisense oligonucleotides, primers, and probes.

[0077] Antisense oligonucleotides, primers, and probes of the inventioncan consist of at least 11, 12, 15, 20, 25, 30, 50, or 100 contiguousnucleotides which are complementary to the coding sequences shown in SEQID NOS:6-10, 18, and 19. A complement of the entire coding sequence canalso be used. Double-stranded subgenomic polynucleotides which compriseall or a portion of the nucleotide sequences shown in SEQ ID NOS:6-10,18, and 19, as well as polynucleotides which encode TNF-L- orTNFR-L-specific antibodies or ribozymes, are also subgenomicpolynucleotides according to the invention.

[0078] Degenerate nucleotide sequences encoding amino acid sequences ofproteins or biologically active protein variants as well as homologousnucleotide sequences which are at least 65%, 75%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleotide sequences shown in SEQ IDNOS.6-10, 18, or 19 are also subgenomic polynucleotides according to theinvention and can be used in the methods disclosed herein. Percentsequence identity between a nucleotide sequence of SEQ ID NOS:6-10, 18or 19 and a putative homologous or degenerate nucleotide sequence isdetermined using computer programs which employ the Smith-Watermanalgorithm, for example as implemented in the MPSRCH program (OxfordMolecular), using an affine gap search with the following parameters: agap open penalty of 12 and a gap extension penalty of 1.

[0079] Nucleotide sequences which hybridize to the coding sequencesshown in SEQ ID NOS:6-10, 18 or 19, or their complements with at most 1,2, 3, 4, 5, 10, 15, 20, 25, 30, or 35% basepair mismatches are alsoTNF-L or TNFR-L subgenomic polynucleotides. For example, using thefollowing wash conditions—2×SSC (0.3 M sodium chloride, 0.03 M sodiumcitrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each;then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, roomtemperature twice, 10 minutes each—homologous TNF-L or TNFR-L sequencescan be identified which contain at most about 25-30% basepair mismatcheswith SEQ ID NOS:6-10, 18 or 19, or their complements More preferably,homologous nucleic acid strands contain 15-25% basepair mismatches, evenmore preferably 5-15% basepair mismatches.

[0080] Species homologs of TNF-L or TNFR-L subgenomic polynucleotides ofthe invention can also be identified by making suitable probes orprimers and screening cDNA expression libraries from other species, suchas mice, monkeys, yeast, or bacteria. It is well known that the T_(m) ofa double-stranded DNA decreases by 1-1.5° C. with every 1% decrease inhomology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Homologous TNF-Lor TNFR-L human polynucleotides or TNF-L or TNFR-L polynucleotides ofother species can therefore be identified, for example, by hybridizing aputative homologous TNF-L or TNFR-L polynucleotide with a polynucleotidehaving a nucleotide sequence of SEQ ID NO:6, 7, 8, 9, 10, 18, or 19,comparing the melting temperature of the test hybrid with the meltingtemperature of a hybrid comprising a polynucleotide having a nucleotidesequence of SEQ ID NOS:6, 7, 8, 9, 10, 18, or 19 and a polynucleotidewhich is perfectly complementary to SEQ ID NO:6, 7, 8, 9, 10, 18, or 19,and calculating the number of basepair mismatches within the testhybrid.

[0081] Nucleotide sequences which hybridize to the coding sequencesshown in SEQ ID NOS:6-10, 18, or 19, or their complements followingstringent hybridization and/or wash conditions are also TNF-L or TNFR-Lsubgenomic polynucleotides. Stringent wash conditions are well known andunderstood in the art and are disclosed, for example, in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed, 1989, at pages9.50-9.51.

[0082] Typically, for stringent hybridization conditions a combinationof temperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a sequence shown in SEQ ID NO:6, 7,8, 9, 10, 18 or 19, and a polynucleotide sequence which is 65%, 75%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, 7, 8, 9,10, 18, or 19 can be calculated, for example, using the equation ofBolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

[0083] T_(m)=81.5° C.-16 6(log₁₀[Na³⁰])+0.41(%G+C)−0.63(%formamide)−600/l), where l=the length of the hybridin basepairs.

[0084] Stringent wash conditions include, for example, 4×SSC at 65° C.,or 50% formamide, 4×SSC at 42° C., or 05×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

[0085] Subgenomic polynucleotides can be isolated and purified free fromother nucleotide sequences using standard nucleic acid purificationtechniques. For example, restriction enzymes and probes can be used toisolate subgenomic polynucleotide fragments which comprise TNF-L orTNFR-L coding sequences. Isolated and purified subgenomicpolynucleotides are in preparations which are free or at least 90% freeof other molecules.

[0086] Complementary DNA (cDNA) molecules which encode TNF-L or TNFR-Lproteins are also TNF-L or TNFR-L subgenomic polynucleotides. cDNAmolecules can be made with standard molecular biology techniques, usingTNF-L or TNFR-L mRNA as a template. cDNA molecules can thereafter bereplicated using molecular biology techniques known in the art anddisclosed in manuals such as Sambrook et al., 1989. An amplificationtechnique, such as the polymerase chain reaction (PCR), can be used toobtain additional copies of subgenomic polynucleotides, using eitherhuman or mammalian genomic DNA or cDNA as a template.

[0087] Alternatively, synthetic chemistry techniques can be used tosynthesize subgenomic polynucleotide molecules The degeneracy of thegenetic code allows alternate nucleotide sequences to be synthesizedwhich will encode a TNF-L protein comprising an amino acid sequenceshown in SEQ ID NOS:1, 2, 3, 4, 5, 17, 20 or a biologically activevariant of one of those sequences.

[0088] The invention also provides polynucleotide probes which can beused to detect TNF-L or TNFR-L sequences, for example, in hybridizationprotocols such as Northern or Southern blotting or in situhybridizations. TNF-L or TNFR-L polynucleotide probes of the inventioncomprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 or morecontiguous nucleotides selected from SEQ ID NOS:6-10, 18, or 19.Polynucleotide probes can comprise a detectable label, such as aradioisotopic, fluorescent, enzymatic, or chemiluminescent label.

[0089] TNF-L or TNFR-L subgenomic polynucleotides can be propagated invectors and cell lines using techniques well known in the art.Expression systems in bacteria include those described in Chang et al,Nature (1978) 275 615, Goeddel et al, Nature (1979) 281: 544, Goeddel etal., Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S. Pat. No.4,551,433, deBoer et al, Proc. Natl. Acad. Sci. USA (1983) 80: 21-25,and Siebenlist et al, Cell (1980) 20: 269.

[0090] Expression systems in yeast include those described in Hinnen etal., Proc. Natl Acad. Sci. USA (1978) 75. 1929; Ito et al., J. Bacteriol(1983) 153: 163; Kurtz et al., Mol. Cell. Biol (1986) 6 142; Kunze etal., J. Basic Microbiol (1985) 25: 14 1; Gleeson et al., J. Gen.Microbiol (1986) 132: 3459, Roggenkamp et al., Mol. Gen. Genet. (1986)202 :302) Das et al., J. Bacteriol. (1984) 158. 1165, De Louvencourt etal., J. Bacteriol (1983) 154: 737, Van den Berg et al., Bio/Technology(1990) 8 135; Kunze et al., J. Basic Microbiol (1985) 25 141; Cregg etal., Mol. Cell. Biol. (1985) 5. 3376, U.S. Pat. Nos. 4,837,148,4,929,555, Beach and Nurse, Nature (1981) 300: 706, Davidow et al.,Curr. Genet. (1985) 10 380, Gaillardin et al., Curr. Genet. (1985) 10:49, Ballance et al., Biochem. Biophy. Res. Commun. (1983) 112 284-289;Tilburn et al, Gene (1983) 26: 205-221, Yelton et al., Proc. Natl Acad.Sci. USA (1984) 81: 1470-1474, Kelly and Hynes, EMBO J (1985) 4: 475479,EP 244,234, and WO 91/00357.

[0091] Expression of TNF-L or TNFR-L subgenomic polynucleotides ininsects can be accomplished as described in U.S. Pat. No. 4,745,051,Friesen et al (1986) “The Regulation of Baculovirus Gene Expression” in:THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed ), EP 127,839,EP 155,476, and Viak et al., J. Gen. Virol. (1988) 69: 765-776, Milleret al., Ann. Rev. Microbiol. (1988) 42: 177, Carbonell et al., Gene(1988) 73: 409, Maeda et al., Nature (1985) 315: 592-594,Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et al.,Proc. Natl. Acad. Sci. USA (1985) 82: 8404, Miyajima et al., Gene (1987)58: 273; and Martin et al., DNA (1988) 7:99. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts are described in Luckow et al, Bio/Technology (1988) 6: 47-55,Miller et al., in GENETIC ENGINEERING (Setlow, J. K. et al. eds.), Vol.8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al., Nature,(1985) 315: 592-594.

[0092] Mammalian expression of TNF-L or TNFR-L subgenomicpolynucleotides can be accomplished as described in Dijkema et al., EMBOJ. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79.6777, Boshart et al., Cell (1985) 41: 521 and U.S. Pat. No. 4,399,216.Other features of mammalian expression can be facilitated as describedin Ham and Wallace, Meth. Enz. (1979) 58: 44, Barnes and Sato, Anal.Biochem. (1980) 102: 255, U.S. Pat. Nos. 4,767,704, 4,657,866,4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. Pat. No. RE30,985.

[0093] TNF-L or TNFR-L subgenomic polynucleotides can be on linear orcircular molecules. They can be on autonomously replicating molecules oron molecules without replication sequences They can be regulated bytheir own or by other regulatory sequences, as is known in the art TNF-Lor TNFR-L subgenomic polynucleotides can be introduced into suitablehost cells using a variety of techniques which are available in the art,such as transferrin-polycation-mediated DNA transfer, transfection withnaked or encapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, and calcium phosphate-mediatedtransfection.

[0094] Polynucleotides of the invention can also be used in genedelivery vehicles, for the purpose of delivering an mRNA oroligonucleotide (either with the sequence of a native mRNA or itscomplement), full-length protein, fusion protein, polypeptide, orribozyme, or single-chain antibody, into a cell, preferably a eukaryoticcell. According to the present invention, a gene delivery vehicle canbe, for example, naked plasmid DNA, a viral expression vector comprisinga polynucleotide of the invention, or a polynucleotide of the inventionin conjunction with a liposome or a condensing agent.

[0095] In one embodiment of the invention, the gene delivery vehiclecomprises a promoter and one of the polynucleotides disclosed herein.Preferred promoters are tissue-specific promoters and promoters whichare activated by cellular proliferation, such as the thymidine kinaseand thymidylate synthase promoters. Other preferred promoters includepromoters which are activatable by infection with a virus, such as theα- and β-interferon promoters, and promoters which are activatable by ahormone, such as estrogen. Other promoters which can be used include theMoloney virus LTR, the CMV promoter, and the mouse albumin promoter.

[0096] A gene delivery vehicle can comprise viral sequences such as aviral origin of replication or packaging signal. These viral sequencescan be selected from viruses such as astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, retrovirus, togavirus or adenovirus. In a preferredembodiment, the gene delivery vehicle is a recombinant retroviralvector. Recombinant retroviruses and various uses thereof have beendescribed in numerous references including, for example, Mann et al.,Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA81:6349, 1984, Miller et al, Human Gene Therapy 1:5-14, 1990, U.S. Pat.Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos WO89/02,468, WO 89/05,349, and WO 90/02,806 Numerous retroviral genedelivery vehicles can be utilized in the present invention, includingfor example those described in EP 0,415,731; WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234, U.S. Pat. No. 5,219,740; WO 9311230; WO9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart,Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al, J.Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP0,345,242 and WO91/02805).

[0097] Particularly preferred retroviruses are derived from retroviruseswhich include avian leukosis virus (ATCC Nos VR-535 and VR-247), bovineleukemia virus (VR-1315), murine leukemia virus (MLV), mink-cellfocus-inducing virus (Koch et al, J. Vir. 49:828, 1984; and Oliff et al,J. Vir. 48:542, 1983), murine sarcoma virus (ATCC Nos. VR-844, 45010 and45016), reticuloendotheliosis virus (ATCC Nos VR-994, VR-770 and 45011),Rous sarcoma virus, Mason-Pfizer monkey virus, baboon endogenous virus,endogenous feline retrovirus (e.g., RD114), and mouse or rat gL30sequences used as a retroviral vector. Particularly preferred strains ofMLV from which recombinant retroviruses can be generated include 4070Aand 1504A (Hartley and Rowe, J. Vir. 19:19, 1976), Abelson (ATCC No.VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al., J. Vir. 67:4722,1993; and Yantchev Neoplasma 26:397, 1979), Gross (ATCC No. VR-590),Kirsten (Albino et al, J. Exp. Med. 164:1710, 1986), Harvey sarcomavirus (Manly et al., J. Vir. 62:3540, 1988; and Albino et al., J. Exp.Med. 164:1710, 1986) and Rauscher (ATCC No. VR-998), and Moloney MLV(ATCC No. VR-190). A particularly preferred non-mouse retrovirus is Roussarcoma virus. Preferred Rous sarcoma viruses include Bratislava (Manlyet al., J. Vir. 62:3540, 1988; and Albino et al, J. Exp. Med. 164:1710,1986), Bryan high titer (e.g. ATCC Nos. VR-334, VR-657, VR-726, VR-659,and VR-728), Bryan standard (ATCC No. VR-140), Carr-Zilber (Adgighitovet al., Neoplasma 27 159, 1980), Engelbreth-Holm (Laurent et al, BiochemBiophys Acta 908 241, 1987), Harris, Prague (e.g., ATCC Nos VR-772, and45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354)viruses.

[0098] Any of the above retroviruses can be readily utilized in order toassemble or construct retroviral gene delivery vehicles given thedisclosure provided herein and standard recombinant techniques (e.g.,Sambrook et al., 1989, and Kunkle, Proc. Natl Acad. Sci. U.S.A. 82.488,1985) known in the art. Portions of retroviral expression vectors can bederived from different retroviruses. For example, retrovector LTRs canbe derived from a murine sarcoma virus, a tRNA binding site from a Roussarcoma virus, a packaging signal from a murine leukemia virus, and anorigin of second strand synthesis from an avian leukosis virus Theserecombinant retroviral vectors can be used to generate transductioncompetent retroviral vector particles by introducing them intoappropriate packaging cell lines (see Ser. No. 07/800,921, filed Nov.29, 1991). Recombinant retroviruses can be produced which direct thesite-specific integration of the recombinant retroviral genome intospecific regions of the host cell DNA. Such site-specific integrationcan be mediated by a chimeric integrase incorporated into the retroviralparticle (see Ser. No. 08/445,466 filed May 22, 1995). It is preferablethat the recombinant viral gene delivery vehicle is areplication-defective recombinant virus.

[0099] Packaging cell lines suitable for use with the above-describedretroviral gene delivery vehicles can be readily prepared (see Ser. No.08/240,030, filed May 9, 1994; see also WO 92/05266) and used to createproducer cell lines (also termed vector cell lines or “VCLs”) forproduction of recombinant viral particles. In particularly preferredembodiments of the present invention, packaging cell lines are made fromhuman (e.g., HT1080 cells) or mink parent cell lines, thereby allowingproduction of recombinant retroviral gene delivery vehicles which arecapable of surviving inactivation in human serum. The construction ofrecombinant retroviral gene delivery vehicles is described in detail inWO 91/02805 These recombinant retroviral gene delivery vehicles can beused to generate transduction competent retroviral particles byintroducing them into appropriate packaging cell lines (see Ser. No.07/800,921). Similarly, adenovirus gene delivery vehicles can also bereadily prepared and utilized given the disclosure provided herein (seealso Berkner, Biotechniques 6:616-627, 1988, and Rosenfeld et al,Science 252-431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282)

[0100] A gene delivery vehicle can also be a recombinant adenoviral genedelivery vehicle. Such vehicles can be readily prepared and utilizedgiven the disclosure provided herein (see Berkner, Biotechniques 6.616,1988, and Rosenfeld et al., Science 252:431, 1991, WO 93/07283, WO93/06223, and WO 93/07282). Adeno-associated viral gene deliveryvehicles can also be constructed and used to deliver proteins orpolynucleotides of the invention to cells in vitro or in vivo The use ofadeno-associated viral gene delivery vehicles in vitro is described inChatterjee et al., Science 258: 1485-1488 (1992), Walsh et al., Proc.Nat'l. Acad. Sci. 89 7257-7261 (1992), Walsh et al., J. Clin. Invest.94: 1440-1448 (1994), Flotte et al, J. Biol. Chem. 268 3781-3790 (1993),Ponnazhagan et al., J. Exp. Med. 179 733-738 (1994), Miller et al.,Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994), Einerhand et al., GeneTher. 2 336-343 (1995), Luo et al, Exp. Hematol. 23: 1261-1267 (1995),and Zhou et al., Gene Therapy 3: 223-229 (1996). In vivo use of thesevehicles is described in Flotte et al., Proc. Nat'l Acad. Sci. 9010613-10617 (1993), and Kaplitt et al., Nature Genet. 8: 148-153 (1994).

[0101] In another embodiment of the invention, a gene delivery vehicleis derived from a togavirus. Preferred togaviruses include alphaviruses,in particular those described in U.S. Ser. No. 08/405,627, filed Mar.15, 1995, WO 95/07994. Alpha viruses, including Sindbis and ELVS virusescan be gene delivery vehicles for polynucleotides of the invention.Alpha viruses are described in WO 94/21792, WO 92/10578 and WO 95/07994.Several different alphavirus gene delivery vehicle systems can beconstructed and used to deliver polynucleotides to a cell according tothe present invention. Representative examples of such systems includethose described in U.S. Pat. Nos. 5,091,309 and 5,217,879 Particularlypreferred alphavirus gene delivery vehicles for use in the presentinvention include those which are described in WO 95/07994, and U.S.Ser. No. 08/405,627.

[0102] Preferably, the recombinant viral vehicle is a recombinantalphavirus viral vehicle based on a Sindbis virus. Sindbis constructs,as well as numerous similar constructs, can be readily preparedessentially as described in U.S. Ser. No. 08/198,450. Sindbis viral genedelivery vehicles typically comprise a 5′ sequence capable of initiatingSindbis virus transcription, a nucleotide sequence encoding Sindbisnon-structural proteins, a viral junction region inactivated so as toprevent fragment transcription, and a Sindbis RNA polymerase recognitionsequence Optionally, the viral junction region can be modified so thatpolynucleotide transcription is reduced, increased, or maintained Aswill be appreciated by those in the art, corresponding regions fromother alphaviruses can be used in place of those described above.

[0103] The viral junction region of an alphavirus-derived gene deliveryvehicle can comprise a first viral junction region which has beeninactivated in order to prevent transcription of the polynucleotide anda second viral junction region which has been modified such thatpolynucleotide transcription is reduced. An alphavirus-derived vehiclecan also include a 5′ promoter capable of initiating synthesis of viralRNA from cDNA and a 3′ sequence which controls transcriptiontermination.

[0104] Other recombinant togaviral gene delivery vehicles which can beutilized in the present invention include those derived from SemlikiForest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370),Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equineencephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCCVR-532), and those described in U.S. Pat. Nos. 5,091,309 and 5,217,879and in WO 92/10578. The Sindbis vehicles described above, as well asnumerous similar constructs, can be readily prepared essentially asdescribed in U.S. Ser. No. 08/198,450.

[0105] Other viral gene delivery vehicles suitable for use in thepresent invention include, for example, those derived from poliovirus(Evans et al., Nature 339:385, 1989, and Sabin et al., J. Biol.Standardization 1:115, 1973) (ATCC VR-58); rhinovirus (Arnold et al., J.Cell. Biochem. L401, 1990) (ATCC VR- 1110); pox viruses, such as canarypox virus or vaccinia virus (Fisher-Hoch et al., PROC. NATL. ACAD. SCI.U.S.A. 86:317, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86, 1989;Flexner et al, Vaccine 8:17, 1990; U.S. Pat. Nos. 4,603,112 and4,769,330; WO 89/01973) (ATCC VR-111, ATCC VR-2010); SV40 (Mulligan etal., Nature 277:108, 1979) (ATCC VR-305), (Madzak et al., J. Gen. Vir.73-1533, 1992); influenza virus (Luytjes et al., Cell 59:1107, 1989;McMicheal et al, The New England Journal of Medicine 309:13, 1983; andYap et a., Nature 273 238, 1978) (ATCC VR-797); parvovirus such asadeno-associated virus (Samulski et al., J. Vir. 63:3822, 1989, andMendelson et al, Virology 166.154, 1988) (ATCC VR-645); herpes simplexvirus (Kit et al., Adv. Exp. Med. Biol. 215:219, 1989) (ATCC VR-977,ATCC VR-260); Nature 277: 108, 1979); human immunodeficiency virus (EPO386,882, Buchschacher et al, J. Vir. 66:2731, 1992); measles virus (EPO440,219) (ATCC VR-24); A (ATCC VR-67; ATCC VR-1247), Aura (ATCC VR-368),Bebaru virus (ATCC VR-600; ATCC VR-1240), Cabassou (ATCC VR-922),Chikungunya virus (ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924),Getah virus (ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927),Mayaro (ATCC VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu(ATCC VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCCVR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCCVR-926), Y-62-33 (ATCC VR-375), O'Nyong virus, Eastern encephalitisvirus (ATCC VR-65; ATCC VR-1242), Western encephalitis virus (ATCCVR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus (Hamreet al., Proc. Soc. Exp. Biol. Med. 121:190, 1966) (ATCC VR-740).

[0106] A polynucleotide of the invention can also be combined with acondensing agent to form a gene delivery vehicle. In a preferredembodiment, the condensing agent is a polycation, such as polylysine,polyarginine, polyornithine; protamine, spermine, spermidine, andputrescine. Many suitable methods for making such linkages are known inthe art (see, for example, Ser. No. 08/366,787, filed Dec. 30, 1994).

[0107] In an alternative embodiment, a polynucleotide is associated witha liposome to form a gene delivery vehicle. Liposomes are small, lipidvesicles comprised of an aqueous compartment enclosed by a lipidbilayer, typically spherical or slightly elongated structures severalhundred Angstroms in diameter. Under appropriate conditions, a liposomecan fuse with the plasma membrane of a cell or with the membrane of anendocytic vesicle within a cell which has internalized the liposome,thereby releasing its contents into the cytoplasm Prior to interactionwith the surface of a cell, however, the liposome membrane acts as arelatively impermeable barrier which sequesters and protects itscontents, for example, from degradative enzymes. Additionally, because aliposome is a synthetic structure, specially designed liposomes can beproduced which incorporate desirable features See Stryer, Biochemistry,pp. 236-240, 1975 (W. H. Freeman, San Francisco, Calif.); Szoka et al.,Biochim. Biophys. Acta 600-1, 1980; Bayer et al., Biochim. Biophys.Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol. 149:119, 1987; Wang etal, PROC NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et al., AnalBiochem. 176:420, 1989, and U.S. Pat. No. 4,762,915. Liposomes canencapsulate a variety of nucleic acid molecules including DNA, RNA,plasmids, and expression constructs comprising polynucleotides suchthose disclosed in the present invention.

[0108] Liposomal preparations for use in the present invention includecationic (positively charged), anionic (negatively charged) and neutralpreparations. Cationic liposomes have been shown to mediateintracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7416, 1987), mRNA (Malone et al., Proc. Natl. Acad.Sci. USA 86:6077-6081, 1989), and purified transcription factors (Debset al., J. Biol. Chem. 265:10189-10192, 1990), in functional form.Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GEBCO BRL, Grand Island,N.Y. See also Feigner et al., Proc. Natl. Acad. Sci. USA 91:5148-5152.87, 1994. Other commercially available liposomes includeTransfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationicliposomes can be prepared from readily available materials usingtechniques well known in the art. See, e.g., Szoka et al., Proc. Natl.Acad. Sct USA 75:4194-4198, 1978; and WO 90/11092 for descriptions ofthe synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes.

[0109] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios Methods for making liposomes using these materialsare well known in the art.

[0110] The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs) Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., METHODS OF IMMUNOLOGY (1983),Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA87:3410-3414, 1990; Papahadjopoulos et al., Biochim. Biophys. Acta394:483, 1975, Wilson et al., Cell 17:77, 1979, Deamer and Bangham,Biochim. Biophys. Acta 443 629, 1976; Ostro et al., Biochem. Biophys.Res. Commun. 76:836 , 1977; Fraley et al., Proc. Natl. Acad. Sci. USA76:3348, 1979; Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA76:145, 1979; Fraley et al., J. Biol. Chem. 255:10431, 1980; Szoka andPapahadjopoulos, Proc. Natl. Acad. Sci. USA 75:145, 1979; andSchaefer-Ridder et al., Science 215:166, 1982.

[0111] In addition, lipoproteins can be included with a polynucleotideof the invention for delivery to a cell. Examples of such lipoproteinsinclude chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, orfusions of these proteins can also be used. Modifications of naturallyoccurring lipoproteins can also be used, such as acetylated LDL. Theselipoproteins can target the delivery of polynucleotides to cellsexpressing lipoprotein receptors. Preferably, if lipoproteins areincluded with a polynucleotide, no other targeting ligand is included inthe composition.

[0112] In another embodiment, naked polynucleotide molecules are used asgene delivery vehicles, as described in WO 90/11092 and U.S. Pat. No.5,580,859. Such gene delivery vehicles can be either DNA or RNA and, incertain embodiments, are linked to killed adenovirus. Curiel et al.,Hum. Gene. Ther. 3:147-154, 1992. Other suitable vehicles includeDNA-ligand (Wu et al., J. Biol. Chem. 264 16985-16987, 1989), lipid-DNAcombinations (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 7417,1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855,1987) and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 882726-2730, 1991).

[0113] One can increase the efficiency of naked polynucleotide uptakeinto cells by coating the polynucleotides onto biodegradable latexbeads. This approach takes advantage of the observation that latexbeads, when incubated with cells in culture, are efficiently transportedand concentrated in the perinuclear region of the cells. The beads willthen be transported into cells when injected into musclePolynucleotide-coated latex beads will be efficiently transported intocells after endocytosis is initiated by the latex beads and thusincrease gene transfer and expression efficiency. This method can beimproved further by treating the beads to increase their hydrophobicity,thereby facilitating the disruption of the endosome and release ofpolynucleotides into the cytoplasm.

[0114] The newly identified receptor proteins play regulatory roles incell proliferation and/or differentiation For example, they can inducethe production of cytokines, immunoglobulins, and cell surface antigensThe receptors can also play a role in the negative regulation ofosteoclastogenesis Soluble TNFR-like receptors can be useful in theneutralization of TNF or TNF-like ligands for the treatment ofrheumatoid arthritis and Crohn's disease. Similarly, restoring normalapoptosis to a cell via these receptors can be used to treat viraldiseases.

[0115] A variety of diseases and conditions can be treated by modulatingthe activity of TNF-L or TNFR-L proteins of the invention. For example,TNFL proteins induce apoptosis of activated T cells, but rescue restingT cells from apoptosis. A TNF-L protein can therefore be used to treatautoimmune diseases, such as myasthenia gravis, insulin-dependentdiabetes mellitus, rheumatoid arthritis, multiple sclerosis, andsystemic lupus erythematosus. TNF-L proteins also have tumor stimulatingproperties Tumors can therefore be treated by inhibiting expression oractivity of a TNF-L protein. Similarly, reducing expression of a TNFR-Lprotein or blocking its ligand binding site can be used to treat tumors,whereas increasing expression of a TNFR-L protein can be used to treatautoimmune diseases such as those disclosed above.

[0116] In one embodiment of the invention, expression of a TNF-L orTNFR-L gene is decreased using a ribozyme, an RNA molecule withcatalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech.1990, Ann. Rev. Biochem. 59:543-568, Cech, 1992, Curr. Opin. Stryct.Biol. 2 605-609; Couture and Stinchcomb, 1996, Trends Genet. 12:510-515. Ribozymes can be used to inhibit gene function by cleaving anRNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat.No. 5,641,673)

[0117] The coding sequences shown in SEQ ID NOS:6-10, 18, and 19 can beused to generate ribozymes which will specifically bind to mRNAtranscribed from a TNF-L or TNFR-L gene. Methods of designing andconstructing ribozymes which can cleave other RNA molecules in trans ina highly sequence specific manner have been developed and described inthe art (see Haseloff et al (1988), Nature 334:585-591). For example,the cleavage activity of ribozymes can be targeted to specific RNAs byengineering a discrete “hybridization” region into the ribozyme. Thehybridization region contains a sequence complementary to the target RNAand thus specifically hybridizes with the target (see, for example,Gerlach et al., EP 321,201). Longer complementary sequences can be usedto increase the affinity of the hybridization sequence for the target.The hybridizing and cleavage regions of the ribozyme can be integrallyrelated; thus, upon hybridizing to the target RNA through thecomplementary regions, the catalytic region of the ribozyme can cleavethe target.

[0118] TNF-L and TNFR-L-specific ribozymes can be introduced into cells,such as neoplastic cells, as part of a DNA construct, as is known in theart. The DNA construct can also include transcriptional regulatoryelements, such as a promoter element, an enhancer or UAS element, and atranscriptional terminator signal, for controlling transcription of theribozyme in the cells.

[0119] Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce the ribozyme-containing DNA construct into cellswhose division it is desired to decrease, as described above.Alternatively, if it is desired that the cells stably retain the DNAconstruct, the DNA construct can be supplied on a plasmid and maintainedas a separate element or integrated into the genome of the cells, as isknown in the art.

[0120] Expression of a TNF-L or TNFR-L gene can also be altered using anantisense oligonucleotide sequence The antisense sequence iscomplementary to at least a portion of the coding sequence of a genehaving a coding sequence shown in SEQ ID NO:6-10, 18, or 19 Preferably,the antisense oligonucleotide sequence is at least six nucleotides inlength, but can be about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides long. Longer sequences can also be used. TNF-L or TNFR-Lantisense oligonucleotide molecules can be provided in a DNA constructand introduced into cells whose division is to be decreased, asdescribed above.

[0121] Antisense oligonucleotides can be composed ofdeoxyribonucleotides, ribonucleotides, or a combination of both.Oligonucleotides can be synthesized manually or by an automatedsynthesizer, by covalently linking the 5′ end of one nucleotide with the3′ end of another nucleotide with non-phosphodiester internucleotidelinkages such alkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters See Brown, 1994, Meth. Mol. Biol 20-1-8; Sonveaux,1994, Meth. Mol. Biol. 26 1-72; Uhlmann et al., 1990, Chem. Rev.90:543-583

[0122] Precise complementarity is not required for successful duplexformation between an antisense molecule and the complementary codingsequence of a TNF-L or TNFR-L gene. Antisense molecules which comprise,for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotideswhich are precisely complementary to a TNF-L or TNFR-L coding sequence,each separated by a stretch of contiguous nucleotides which are notcomplementary to adjacent TNF-L or TNFR-L coding sequences, can providetargeting specificity for TNF-L or TNFR-L mRNA. Preferably, each stretchof contiguous nucleotides is at least 4, 5, 6, 7, or 8 or morenucleotides in length. Non-complementary intervening sequences arepreferably 1, 2, 3, or 4 nucleotides in length. One skilled in the artcan easily use the calculated melting point of an antisense-sense pairto determine the degree of mismatching which will be tolerated between aparticular antisense oligonucleotide and a particular TNF-L or TNFR-Lcoding sequence.

[0123] TNF-L or TNFR-L antisense oligonucleotides can be modifiedwithout affecting their ability to hybridize to a TNF-L or TNFR-L codingsequence. These modifications can be internal or at one or both ends ofthe antisense molecule. For example, internucleoside phosphate linkagescan be modified by adding cholesteryl or diamine moieties with varyingnumbers of carbon residues between the amino groups and terminal ribose.Modified bases and/or sugars, such as arabinose instead of ribose, or a3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the5′ phosphate group are substituted, can also be employed in a modifiedantisense oligonucleotide. These modified oligonucleotides can beprepared by methods well known in the art. Agrawal et al., 1992, TrendsBiotechnol. 10:152-158; Uhlmann et al., 1990, Chem. Rev. 90:543-584;Uhlmann et al., 1987, Tetrahedron. Lett. 215:3539-3542.

[0124] Antibodies which specifically bind to a TNF-L and TNFR-L proteincan also be used to alter effective levels of TNF-L or TNFR-L geneexpression. TNF-L and TNFR-L-specific antibodies bind to TNF-L andTNFR-L proteins and prevent the proteins from functioning in the cellConstruction of such antibodies is disclosed above.

[0125] Expression of an endogenous TNF-L or TNFR-L gene in a cell canalso be altered by introducing in frame with the endogenous 7NF-L orTNFR-L gene a DNA construct comprising a TNF-L or TNFR-L targetingsequence, a regulatory sequence, an exon, and an unpaired splice donorsite by homologous recombination, such that a homologously recombinantcell comprising the DNA construct is formed. The new transcription unitcan be used to turn the TNF-L or TNFR-L gene on or off as desired. Thismethod of affecting endogenous gene expression is taught in U.S. Pat.No. 5,641,670.

[0126] The targeting sequence is a segment of at least 10, 12, 15, 20,or 50 contiguous nucleotides selected from a nucleotide sequence shownin SEQ ID NO:6-10, 18, or 19. The transcription unit is located upstreamof a coding sequence of the endogenous TNF-L or TNFR-L gene. Theexogenous regulatory sequence directs transcription of the codingsequence of the TNF-L or TNFR-L gene.

[0127] Preferably, the mechanism used to decrease expression of theTNF-L or TNFR-L gene, whether ribozyme, antisense nucleotide sequence,or antibody, decreases expression of the gene by 50%, 60%, 70%, or 80%Most preferably, expression of the gene is decreased by 90%, 95%, 99%,or 100%. The effectiveness of the mechanism chosen to alter expressionof the gene can be assessed using methods well known in the art, such ashybridization of nucleotide probes to mRNA of the gene, quantitativeRT-PCR, or detection of a TNF-L and TNFR-L protein using specificantibodies of the invention.

[0128] TNF-L and TNFR-L proteins or subgenomic polynucleotides can beused in therapeutic compositions for treating a variety of TNF-mediateddisorders. Therapeutic compositions of the invention which compriseTNF-L protein or TNF-L protein encoding polynucleotides can be used, forexample, to treat disorders in which abnormal numbers of T cells becomeactivated. Activated T-lymphocytes are associated with disease in graftversus host reactions (e.g., bone marrow transplantation) and most formsof autoimmunity, including but not restricted to, multiple sclerosis,rheumatoid arthritis, lupus, and myasthenia gravis.T-lymphocyte-mediated primary diseases, such as juvenile diabetes, canalso be treated using TNF-L protein or protein encoding polynucleotides.

[0129] TNF-L and TNFR-L therapeutic compositions of the invention cancomprise a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known to those in the art. Such carriersinclude, but are not limited to, large, slowly metabolizedmacromolecules, such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Pharmaceutically acceptable salts can also beused in the composition, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates, or sulfates, as well as thesalts of organic acids such as acetates, proprionates, malonates, orbenzoates.

[0130] TNF-L or TNFR-L therapeutic compositions can also containliquids, such as water, saline, glycerol, and ethanol, as well assubstances such as wetting agents, emulsifying agents, or pH bufferingagents. Liposomes, such as those described in U.S. Pat. No. 5,422,120,WO 95/13796, WO 91/14445, or EP 524,968 B1, can also be used as acarrier for a therapeutic TNF-L or TNFR-L composition.

[0131] Typically, a therapeutic TNF-L or TNFR-L composition is preparedas an injectable, either as a liquid solution or suspension; however,solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection can also be prepared. A TNF-L or TNFR-L compositioncan also be formulated into an enteric coated tablet or gel capsuleaccording to known methods in the art, such as those described in U.S.Pat. No. 4,853,230, EP 225,189, AU 9,224,296, and AU 9,230,801.

[0132] Proliferative disorders, such as neoplasias, dysplasias, andhyperplasias, can be treated by administration of a therapeutic TNF-Land TNFR-L composition which will inhibit TNF-L activity or expressionNeoplasias which can be treated with the therapeutic compositioninclude, but are not limited to, melanomas, squamous cell carcinomas,adenocarcinomas, hepatocellular carcinomas, renal cell carcinomas,sarcomas, myosarcomas, non-small cell lung carcinomas, leukemias,lymphomas, osteosarcomas, central nervous system tumors such as gliomas,astrocytomas, oligodendrogliomas, and neuroblastomas, tumors of mixedorigin, such as Wilms' tumor and teratocarcinomas, and metastatictumors.

[0133] Proliferative disorders which can be treated with a therapeuticTNF-L composition include disorders such as anhydric hereditaryectodermal dysplasia, congenital alveolar dysplasia, epithelialdysplasia of the cervix, fibrous dysplasia of bone, and mammarydysplasia Hyperplasias, for example, endometrial, adrenal, breast,prostate, or thyroid hyperplasias, or pseudoepitheliomatous hyperplasiaof the skin can be treated with TNF-L or TNFR-L therapeuticcompositions.

[0134] Even in disorders in which TNF-L or TNFR-L mutations are notimplicated, decreasing expression of a TNF-L gene or a TNFR-L gene ordecreasing a TNF-L or TNFR-L protein function can have a therapeuticapplication. In these disorders, decreasing TNF-L or TNFR-L expressionor function can help to suppress tumors. Similarly, in tumors in whichTNF-L or TNFR-L expression is not aberrant, effecting TNF-L or TNFR-Ldownregulation or decrease of TNF-L or TNFR-L activity can suppressmetastases.

[0135] Administration of therapeutic compositions of the invention caninclude local or systemic administration, including injection, oraladministration, particle gun, or catheterized administration, andtopical administration. Various methods can be used to administer atherapeutic composition directly to a specific site in the body. Forexample, a small metastatic lesion can be located and a therapeuticcomposition injected several times in several different locations withinthe body of tumor Alternatively, arteries which serve a tumor can beidentified, and a therapeutic composition injected into such an artery,in order to deliver the composition directly into the tumor.

[0136] A tumor which has a necrotic center can be aspirated and thecomposition injected directly into the now empty center of the tumor Atherapeutic composition can be directly administered to the surface of atumor, for example, by topical application of the composition X-rayimaging can be used to assist in certain of the above delivery methods.Combination therapeutic agents, such as an anti-TNF-L neutralizingantibody and another therapeutic agent, can be administeredsimultaneously or sequentially.

[0137] Alternatively, a therapeutic composition can be introduced intohuman cells ex vivo, and the cells then replaced into the human. Cellscan be removed from a variety of locations including, for example, froma selected tumor or from an affected organ. In addition, the therapeuticcomposition can be inserted into non-tumorigenic cells, for example,dermal fibroblasts or peripheral blood leukocytes. If desired,particular fractions of cells such as a T cell subset or stem cells canalso be specifically removed from the blood (see, for example, PCT WO91/16116). The removed cells can then be contacted with the therapeuticcomposition utilizing any of the above-described techniques, followed bythe return of the cells to the human, preferably to or within thevicinity of a tumor. The above-described methods can additionallycomprise the steps of depleting fibroblasts or other non-contaminatingtumor cells subsequent to removing tumor cells from a human, and/or thestep of inactivating the cells, for example, by irradiation.

[0138] Receptor-mediated targeted delivery of therapeutic compositionscontaining TNF-L or TNFR-L subgenomic polynucleotides to specifictissues can also be used. Receptor-mediated DNA delivery techniques aredescribed in, for example, Findeis et al. (1993), Trends in Biotechnol.11, 202-05; Chiou et al (1994), GENE THERAPEUTICS: METHODS ANDAPPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.); Wu & Wu (1988),J. Biol. Chem. 263, 621-24; Wu et al. (1994), J. Biol Chem. 269, 542-46;Zenke et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59; Wu etal. (1991), J. Biol. Chem. 266, 338-42.

[0139] Both the dose of the TNF-L or TNFR-L composition and the means ofadministration can be determined based on the specific qualities of thetherapeutic composition, the condition, age, and weight of the patient,the progression of the disease, and other relevant factors If thecomposition contains TNF-L or TNFR-L protein, polypeptide, or antibody,effective dosages of the composition are in the range of about 5 μg toabout 50 μg/kg of patient body weight, about 50 μg to about 5 μg/kg,about 100 μg to about 500 μg/kg of patient body weight, and about 200 toabout 250 μg/kg.

[0140] Therapeutic compositions containing TNF-L or TNFR-L subgenomicpolynucleotides, ribozymes, or antisense oligonucleotides can beadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol. Concentration ranges of about500 ng to about 50 mg, about 1 μg to about 2 μg, about 5 μg to about 500μg, and about 20 μg to about 100 μg of DNA can also be used during agene therapy protocol. Factors such as method of action and efficacy oftransformation and expression are considerations that will effect thedosage required for ultimate efficacy of the TNF-L or TNFR-L subgenomicpolynucleotides.

[0141] Where greater expression is desired over a larger area of tissue,larger amounts of a TNF-L or TNFR-L therapeutic composition or the sameamount readministered in a successive protocol of administrations, orseveral administrations to different adjacent or close tissue portionsof for example, a tumor site, may be required to effect a positivetherapeutic outcome. In all cases, routine experimentation in clinicaltrials will determine specific ranges for optimal therapeutic effect.

[0142] The invention provides knock-out mammals whose endogenous TNF-Lor TNFR-L gene is not expressed Methods of making knock-out mammals arewell known in the art. The mammal can be any experimental mammal, suchas a mouse, rat, or rabbit; however, a mouse is preferred The endogenouswild-type TNF-L or TNFR-L gene of the mammal can be deleted entirely,resulting in an absence of TNF-L or TNFR-L protein in the mammal.Alternatively, mutations such as deletions, insertions, missensesubstitutions, or inversions, can be introduced into a TNF-L or TNFR-Lgene. Such mutations result in expression of truncated or otherwiseaberrant forms of TNF-L or TNFR-L protein in the knock-out mammalMammalian cell lines which do not express an endogenous TNF-L or TNFR-Lgene can also be constructed, as is known in the art.

[0143] Knock-out mammals and cells of the invention are useful as modelsystems for studying the effects of drugs in the absence of wild-typeTNF-L or TNFR-L protein or in the presence of altered forms of the TNF-Lor TNFR-L protein in the mammal or cell. Knock-out mammals can also beused to develop therapeutic treatments for diseases associated withalterations in TNF-L or TNFR-L gene expression, such as neoplasia orvarious autoimmune diseases.

[0144] The invention also provides screening methods which can be usedto identify chemical agents which may have use in therapy, for example,regulators of the disclosed genes and proteins can be screened using avariety of methods. These include ligand binding (Zhang et al., J. Biol.Chem 267:24069-24075), cytotoxicity (Creasey, Cancer Res. 47:145-149,1987, Geigert, Develop. Biol. Standard 69.129; Tsujimoto, J. Biochem.101:919-925, 1987; Kamijo, Biochem. Biophys. Res. Commun. 160:830-825,1989; Sidhu, Anticancer Res. 9:1569-1576, 1989), differentiation(Kamijo, 1989), maturation of osteoclasts from hematopoietic precursors(Lacey, Endocrinology 136:2367-2376, 1995), and proliferation(Tsujimoto, 1989).

[0145] The ability of a test compound or a potential therapeutic agentto stimulate or inhibit activity of a TNF-L or TNFR-L protein can beassessed by determining or measuring the viability of the population ofcells A test compound which increases or decreases cell lysis or celldeath is a modulator of the TNF-L or TNF-LR protein and can be used as atherapeutic agent to regulate TNF activities, such as cell lysis or celldeath. A test compound which increases cell lysis or cell death may beparticularly useful in treatment of neoplastic growth. The polypeptideof the invention can be applied to the cell exogenously, or it can beexpressed by a cell which has been transfected with a subgenomicpolynucleotide encoding the polypeptide.

[0146] Methods for measuring the viability of cells can be any which areknown in the art. Cell death can be determined by contacting the cellwith a dye and viewing it under a microscope Viable cells can beobserved to have an intact membrane and do not stain, whereas dying ordead cells having “leaky” membranes do stain. Incorporation of the dyeby the cell indicates the death of the cell The most common dye used inthe art for this purpose is trypan blue. Viability of cells can also bedetermined by detecting DNA synthesis. Cells can be cultured in cellmedium with labeled nucleotides, such as [³H]-thymidine. The uptake orincorporation of the labeled nucleotides by cells indicates DNAsynthesis and cell viability. Death of tumor cells in vivo can bemonitored by observing regression or shrinkage of a tumor. Any suitablediagnostic technique can be applied.

[0147] Other cellular proteins which are involved in the same biologicalpathways can be identified by looking for proteins which interact withthe disclosed polypeptides. Natural ligands can therefore be identifiedfor the receptor proteins, and natural receptor proteins can beidentified for the ligands. Complex formation can be detected in vitroor in vivo. Many methods for detecting formation of protein complexesare known in the art, and any such methods can be used. For example, theyeast two-hybrid system can be used in cells to detect proteins whichinteract with the disclosed ligands and receptors. Alternatively,protein complex formation can be tested in vitro and complexes detectedby altered mobility on non-denaturing gels, or byco-immunoprecipitation.

[0148] Expression of TNFR-L proteins can serve as a marker of neoplasia.TNFR-L proteins can be detected in body samples, including tissues,serum, urine, sputum, and feces, using immunological techniques.Expression can also be observed by measuring or detecting mRNA encodingthe receptors. Any suitable technique can be used including but notlimited to Northern blotting and RT-PCR.

[0149] A TNF-R or TNFR-L subgenomic polynucleotide can also be deliveredto subjects for the purpose of screening test compounds for those whichare useful for enhancing transfer of TNF-L or TNFR-L subgenomicpolynucleotides to the cell or for enhancing subsequent biologicaleffects of TNF-L or TNFR-L subgenomic polynucleotides within the cellSuch biological effects include hybridization to complementary TNF-L orTNFR-L mRNA and inhibition of its translation, expression of a TNF-L orTNFR-L subgenomic polynucleotide to form a TNF-L or TNFR-L mRNA,single-chain antibody, ribozyme, oligonucleotide, or protein and/orreplication and integration of a TNF-L or TNFR-L subgenomicpolynucleotide. The subject can be a cell culture or an animal,preferably a mammal more preferably a human.

[0150] Test compounds which can be screened include any substances,whether natural products or synthetic, which can be administered to thesubject in vitro or in vivo Libraries or mixtures of test compounds canbe tested The test compound can be a pharmacologic agent already knownin the art or can be a compound previously unknown to have anypharmacological activity. The test compound can be naturally occurringor designed in the laboratory. It can be isolated from microorganisms,animals, or plants, and can be produced recombinantly, or synthesized bychemical methods known in the art. Test compounds or substances can bedelivered before, after, or concomitantly with a TNF-L or TNFR-Lsubgenomic polynucleotide. They can be administered separately or inadmixture with a TNF-L or TNFR-L subgenomic polynucleotide.

[0151] Integration of a delivered TNF-L or TNFR-L subgenomicpolynucleotide can be monitored by any means known in the art. Forexample, Southern blotting of the delivered TNF-L or TNFR-L subgenomicpolynucleotide can be performed. A change in the size of the fragmentsof a delivered polynucleotide indicates integration. Replication of adelivered polynucleotide can be monitored inter alia by detectingincorporation of labeled nucleotides combined with hybridization to aTNF-L or TNFR-L probe. Expression of a TNF-L or TNFR-L subgenomicpolynucleotide can be monitored by detecting production of TNF-L orTNFR-L mRNA which hybridizes to the delivered polynucleotide or bydetecting TNF-L or TNFR-L protein. TNF-L or TNFR-L protein can bedetected immunologically. Thus, the delivery of TNF-L or TNFR-Lsubgenomic polynucleotides according to the present invention providesan excellent system for screening test compounds for their ability toenhance transfer of TNF-L or TNFR-L polynucleotides to a cell, byenhancing delivery, integration, hybridization, expression, replicationor integration in a cell vitro or in vivo in an animal, preferably amammal, more preferably a human.

[0152] The TNFL1 gene (SEQ ID NO:6) maps to human chromosome 13q34.Polynucleotide probes of TNFL1 can therefore be used to identify thisregion of chromosome 13 in metaphase spreads of human chromosomes.Preparations of human metaphase chromosomes can be prepared usingstandard cytogenetic techniques from human primary tissues or celllines. Polynucleotide probes comprising at least 12 contiguousnucleotides selected from the nucleotide sequence shown in SEQ ID NO:6are used to identify the human chromosome The polynucleotide probes canbe labeled, for example, with a radioactive, fluorescent, biotinylated,or chemiluminescent label, and detected by well known methodsappropriate for the particular label selected. Protocols for hybridizingpolynucleotide probes to preparations of metaphase chromosomes are alsowell known in the art. A polynucleotide probe will hybridizespecifically to nucleotide sequences in the chromosome preparationswhich are complementary to the nucleotide sequence of the probe.

[0153] A polynucleotide probe which hybridizes specifically to humanchromosome region 13q34 hybridizes to nucleotide sequences present inthe TNFL1 gene and not to nucleotide sequences present in other humangenes. A polynucleotide probe which hybridizes specifically to an TNFL1gene provides a detection signal at least 5-, 10-, or 20-fold higherthan the background hybridization provided with non-TNFL1 codingsequences.

[0154] A human chromosome which specifically hybridizes to an TNFL1polynucleotide probe is identified as a human chromosome 13. Preferably,the polynucleotide probe identifies the long arm of human chromosome 13.More preferably, the polynucleotide probe identifies a q34 region ofhuman chromosome 13.

[0155] The complete contents of the references cited in this disclosureare expressly incorporated by reference herein The following examplesare illustrative and are not meant to limit the scope of the inventiondisclosed herein.

EXAMPLE 1

[0156] This example describes cloning of the fill-length cDNA for TNFL1.

[0157] TNFL1 was first identified from a database of expressed sequencetags (ESTs) by its homology to other members of the TNF family. Thefull-length cDNA was isolated by screening a liver cDNA library applyingthe genetrapper technique (Gibco). A liver library from Gibco BRL wasscreened using the Genetrapper cDNA positive selection system (catalogno 10356-020) and two oligonucleotide primers. The sequence of thebiotinylated primer is: 5′AGGTCCATGTCTTTGGG3′ (SEQ ID NO:11) thesequence of the non biotinylated primer is: 5′GGGGATGAATTGAGTCTG3′ (SEQID NO:12). The product of the repair reaction was transformed, plated onLB+ Amp (100 μg/ml) plates. The colonies were analyzed by colonyhybridization with a radioactive fragment prepared by PCR using theprimers 5′GTGCCCTCGAAGAAAAAG3′ (SEQ ID NO:13) and 5′GCAAGTTGGAGTTCATC3′(SEQ ID NO:14).

[0158] The longest open reading frame was 1280 bp long and contained apoly A tail as well as an in-frame stop codon at position −117 upstreamof the ATG at position +1, suggesting that this clone was full-length.The nucleotide sequence surrounding this ATG also matched the Kozakconsensus sequence. The open reading frame encodes a protein of 285amino acids which we named Tumor Necrosis Factor Like 1 (TNFL1 ) (FIG.1A).

[0159] The lack of a signal sequence at the N-terminus and the presenceof an internal hydrophobic domain are indicative of a type IItransmembrane structure, which is similar to the structure of most ofthe other members of the TNF family with the exception of lymphotoxin a.Two potential N-glycosylation sites were also identified in theextracellular region of the protein. When aligned with theextra-cellular domains from other members of the TNF family, theextracellular domain of the TNFL1 protein showed an overall homology of28% to the proteolytically cleaved form of TNF (FIG. 1B).

EXAMPLE 2

[0160] This example shows the tissue distribution of TNFL1 mRNA.

[0161] Northern blots showing mRNAs from different tissues and cancerouscell lines were purchased from Clontech. A Northern blot with mRNAs fromhematopoietic cell lines and various cell types of the immune system wasprepared with 2 μg of poly A mRNA.

[0162] A probe prepared by digestion of the TNFL1 cDNA with EcoRI andXhoI was labeled by random priming with ³⁵S and Klenow enzyme (Rediprimekit from Amersham). The hybridization was performed in the Expresshybbuffer purchased from Clontech.

[0163] A 3 kb messenger mRNA corresponding to TNFL1 mRNA was detectedmainly in the organs of the immune system, such as peripheral bloodlymphocytes, spleen, and thymus, as well as in the small intestine andovary (FIG. 2A) Human TNFL1 mRNA was also detected in a few human cancercell lines such as the chronic myelogenous leukemia cell line K562 andthe melanoma cell line G361 (FIG. 2A). Mouse mRNA was detected in heart,spleen, and lung using as a probe a mouse EST sequence which ishomologous to the human TNFL1 sequence (Accession No. AA254417) (FIG.2B).

[0164] Because TNFL1 mRNA was expressed in the spleen of both mouse andhuman samples as well as in peripheral blood leukocytes, a more preciseanalysis of the protein expression levels was carried out in the sametissues.

EXAMPLE 3

[0165] This example demonstrates expression of protein levels in mouseand human tissues.

[0166] A polyclonal antibody (D2710) was raised against amino acids234-248 of TNFL1 and purified on a protein G column followed by apeptide affinity column. Amino acids 234-248 are highly conservedbetween the human and the mouse protein and differ by only 4 aminoacids.

[0167] This antibody was able to recognize a purified 30 kDa TNFL1protein by Western blot analysis (FIG. 3A, lane 5). A single bandcorresponding to a 45 kDa protein was detected in cytoplasmic extractsfrom mouse bone marrow-derived dendritic cells and human monocytes. Boththe 30 kDa and the 45 kDa bands were absent after incubation of D2710with an excess of competitor peptide (FIG. 3A, lanes 2, 4, and 6),suggesting that the 45 kDa protein corresponds to the full-length TNFL1protein.

[0168] The affinity purified antibody was also able to detect TNFL1expressed in insect cells. Insect cells were infected with a recombinantbaculovirus expressing the TNFL1 protein and analyzed by flow cytometry.The protein TNFL1 was detected by intracellular staining with D2710after fixation and permeabilization of the cells infected with therecombinant virus but not the wild type virus (FIG. 3B).

[0169] Immunohistochemistry experiments were performed on sections frommouse spleen and lymphoid organs using the polyclonal antibody D2710.The spleen was isolated from an animal perfused with 4% paraformaldehydein PBS, incubated in the same solution for one additional hour, andincubated overnight at 4° C. in a 10% sucrose solution. The spleen wasthen embedded in OCT prior to cryo-sectioning. The sections were storedat −80° C. Immunostaining was carried out using the following protocol.The sections were blocked in normal donkey IgG (whole molecule H+L;Jackson 017 000 003; lot 39113 at 25 5 g/l) diluted 1·100 in 1×PBS andFc block diluted 1:50 (Pharmingen, catalog no. 0124A). The sections werethen incubated with primary antibodies D2710, anti-mouse CD11c (HL3,Pharmingen, catalog no 09702D), anti-mouse Th1.2 CD90.2 (53-2.1,Pharmingen, catalog no. 01122A), or anti-mouse CD45R/B220 (RA3-6B2,Pharmingen, catalog no. 01122A) diluted 1:50 in blocking reagent. Thesections were washed three times for 3 minutes each in PBS.

[0170] The sections were then incubated in secondary antibody(biotin-labeled donkey anti-rabbit F(ab)₂ (Jackson 711-066-152) orbiotin-labeled donkey anti-rat F(ab′)₂(Jackson 721-066-153) diluted1:100 in PBS, washed 3 times for 3 min in PBS, and incubated for 30minutes at room temperature with pre-equilibrated ABC-AlkalinePhosphatase reagent (Vector) or ABC-peroxidase (Vector). The sectionswere again washed 3 times for 3 minutes in PBS and incubated in a colordeveloping reaction with levamisole (Vector; SK 5000) using a Vectorblack AP substrate kit (SK5200), Vector red AP substrate kit (SK 5100),or Vector AEC peroxidase substrate kit (SK4200). After washing again inPBS, alkaline phosphatase stained sections were counterstained withhematoxylin nuclear counterstain (Vector; H3401) and methyl green(Vector; H3402). Other sections were mounted in fluoromount (SouthernBiotechnology Associates, catalog no 100-01, Fisher OB100-01).

[0171] Normal rabbit IgG (R& D Systems, catalog no. AB-105C) at a 1:50dilution, secondary antibody at a dilution of 1.100, and secondaryantibody alone at a dilution of 1.100 were used as negative controls.

[0172] TNFL1 was constitutively and specifically expressed as a cellsurface-bound protein in normal spleen (FIG. 3C), but was weaklyexpressed in lymph nodes, mesenteric lymph nodes, and Peyer's patches.In the spleen, the pattern of expression was restricted to the marginalzone and the red pulp. The region stained with monoclonal antibodiesdirected against markers of the T cell population and of the B cell zone(Th1-2 and B220 respectively) did not overlap with the region stainedwith the antibody specific for TNFL1 (FIG. 3D) A monoclonal antibodydirected against the dendritic cell marker CD11c stained the T cell areaand the marginal zone, as well as some isolated cell in the red pulp(FIG. 3D)

[0173] Although it is possible that some cell sub-types present both theCD11c antigen and the TNFL1 protein at their surface, TNFL1 does notseem to be an exclusive marker of dendritic cells in the spleen TheMac-3 antigen, a marker for macrophages and monocytes mainly localizedin the red pulp, showed a pattern of expression very similar to the oneobserved with TNFL1 (FIG. 3D). Overall, these results suggest that TNFL1is expressed at the surface of splenic macrophages, monocytes, ordendritic cells usually present in the marginal zone and the red pulp.

[0174] Flow cytometry experiments were performed on human PBMCs isolatedfrom whole blood by Ficoll gradient centrifugation and the PBMCsanalyzed. TNFL1 was found to be constitutively expressed on monocytesand B cells, but not on resting CD4+ and CD8+ T cells. Mouse bonemarrow-derived dendritic cells cultivated for 10 days in the presence ofGM-CSF also showed some surface staining with the TNFL1 antibody (FIG.4A). After incubation of PBMCs with anti-CD3 and anti-CD28 antibodiesfor 6 days in the presence of IL2, TNFL1 was shown to be upregulated atthe surface of T cells (FIG. 4B).

EXAMPLE 4

[0175] This example demonstrates expression of a soluble form of TNFL1in the periplasm of E. coli.

[0176] A chimeric soluble version of TNFL1 was expressed in E. coli as afusion protein comprising the extracellular portion of TNFL1 (aminoacids 113-285; FIG. 5A) and the pelB signal sequence for periplasmiclocalization. The EYMPMD peptide (SEQ ID NO:15) was inserted between thesignal sequence and the TNFL1 sequence for convenient affinitypurification. The cDNA for TNFL1 was cloned into the vector pET-22b(+)from Invitrogen, which contains the pelB signal sequence for periplasmiclocalization. A 100 ml culture was grown at 37° C. until it reached anOD of 0.7-0.9 and then grown at 25° C. for 24 hours after induction by 1mM IPTG. The pellet was centrifuged at 4000×g for 10 minutes andresuspended in 10 ml 30 mM Tris HCl, 20% sucrose, pH 8.0. After additionof 1 mM EDTA, the sample was incubated at room temperature for 5-10 min.The sample was then centrifuged at 8000×g at 4° C. for 10 min. Thesupernatant was removed, and the pellet was resuspended in 10 mlice-cold 5 mM MgSO₄. After a 10 minute incubation in an ice/water bath,the sample was centrifuged at 8000×g at 4° C. for 10 min. Thesupernatant containing the periplasmic fraction was stored at −80° C. in15% glycerol. A control sample was processed in a similar way with anempty vector construct.

[0177] A similar fusion construct was also designed for expression andpurification from COS cells using the pSecTag vector (Invitrogen) with asignal sequence from the mouse Ig e chain.

EXAMPLE 5

[0178] This example demonstrates purification of the TNFL1 fusionprotein from E. coli.

[0179] BL21 (DE3) transformed E. coli were grown in a 10-1 fermenter toan OD of 29-31 before induction with IPTG The cells were harvested in aBeckman J-6B centrifuge. The wet cell paste was subjected to osmoticshock treatment for periplasmic extraction (lot 10229-142) orlysozyme/EDTA spheroplast formation with retention of the spheroplastsupernatant as the periplasmic fraction (lot 981001-M8). Using a PallFiltron Centrasette tangential flow apparatus and 2 Centrasette 10 kDNMWCO membranes, the resulting periplasmic fraction was concentrated to1 liter, then buffer-exchanged by constant-flow diafiltration in thesame apparatus against at least 6 volumes of PBS. The resulting solutionwas centrifuged at 10,000 rpm at 4° C. for 50 minutes in a Beckman J2-21centrifuge with a JA-10 rotor

[0180] The resulting supernatant was precipitated with 50% ammoniumsulfate using an equal volume of saturated ammonium sulfate. Theresulting pellet was resuspended in ¼ the original volume of PBS andloaded onto a glu-tag monoclonal antibody affinity column at a flow rateof 30 cm/hr. Following the load, a wash of 5-10 CV of PBS +0.2% Tween 20was performed, followed by 2 CV of PBS. Elution was effected by 5 CV of0.1 mg/ml EYMPTD peptide (SEQ ID NO. 16) (Research Genetics) followed byPBS. A strip of 1.5 CV 0.1 M glycine pH 2.7 was also collected intoone-tenth volume of 1 M Tris pH 8, and the column was neutralized with 1CV of the same.

[0181] The peptide elution was concentrated in an Amicon 8400stirred-cell with a YM-10 membrane. Peptide removal was effected byovernight dialysis of the concentrate against PBS (except for thesamples treated to remove residual detergent). Endotoxin removal was byTriton X-114 phase separation. One-ninth volume of 10% Triton X-114protein grade (Calbiochem) was added to samples chilled on ice. Constantagitation for 30 min. at 4° C. was followed by 15 min. incubation in a37° C. water bath. Centrifugation at 10,000×g recovered the supernatantas the detergent-poor aqueous fraction. The cycle was repeated 3-5times, resulting in endotoxin levels of less than or equal to 0.05EU/ml.

[0182] Detergent was removed by an anion-exchange chromatography step inwhich the protein was bound and eluted while the detergent and elutionpeptide flowed through. A Pharmacia HR 10/5 column packed with 1.7 mlTMAE-Fractogel (EM Merck) was equilibrated in 25 mM NaPi, pH 7.4, at 1ml/minute on an Akta Explorer FPLC system. Fractions of 1 ml werecollected across a 15 CV gradient from 0-0 6 M NaCl in 25 mM NaPi, pH 74. The protein eluted at a conductivity of about 22 mS/cm. The peakfractions were pooled and concentrated, if necessary, in a AmiconCentriplus-10 centrifugal concentration device.

[0183] The purified protein was detected in a Western blot with both thetag antibody and the D2710 antibody as a 30 kD protein (FIG. 3A). Aminor additional band at 21 kD was also observed. This band wasidentified by microsequencing as a degradation product of the 30 kDprotein (FIG. 5A)

EXAMPLE 6

[0184] This example demonstrates the effects of TNFL1 on T and B cells.

[0185] T cells isolated from healthy donors were activated for two daysin the presence of anti-CD3 and anti-CD28 antibodies and incubated foran additional two days in the presence of different concentrations ofpurified TNFL1. Mouse and human T cells were purified by negativeselection from PBMCs on a mouse or human T cell enrichment column (R&DSystems), and B cells were isolated by positive selection on DynabeadsM-450 Pan-B and detached from the beads with the polyclonal antibodyDetachabead from Dynal. The T cells were activated for 2 days withanti-CD3 and anti-CD28 antibodies at a concentration of 10 ig/ml.

[0186] T cell proliferation was assessed by thymidine incorporation atday 5. The assays were performed in a 96 well plate format with 100,000cells/well. The thymidine incorporation assay was performed by additionof ³H-thymidine at a concentration of 1 mCi/well. After 8 hours, thecells were harvested onto a filter-paper and ³H-thymidine uptake wasmeasured by liquid scintillation counting.

[0187] The dose-response curve shows a 30-fold decrease in thymidineincorporation at the highest concentrations of TNFL1 (FIG. 5B, left).Resting T cells incubated with the ligand for two days did not show anydecrease in thymidine incorporation (FIG. 5B, right).

[0188] In a separate experiment, the effect of TNFL1 was tested onanti-CD3- and anti-CD28-activated T cells and on anti-CD40-activated Bcells. The T cells were activated for 2 days with anti-CD3 and anti-CD28antibodies at a concentration of 10 μg/ml, and the B cells wereactivated for 2 days in the presence of anti-CD40 antibody at aconcentration of 10 μg/ml. Both cell types showed a strong andcomparable decrease in thymidine incorporation after addition of TNFL1(FIG. 5C) TNFL1 also inhibited the proliferation of murine T cellsactivated by allogeneic bone marrow-derived DCs by 90%.

[0189] We also tested the ability of TNFL1 to decrease the proliferationrate of mouse T cells activated by allogeneic bone marrow-deriveddendritic cells in a mixed lymphocyte reaction (MLR) For the MLRreaction, 100,000 mouse T cells were mixed with 10,000 mouse dendriticcells Dendritic cells were prepared from red blood cell-depleted bonemarrow cells. The cells were resuspended in culture medium in abacterial Petri dish at a concentration of 0.2×10⁶ cells per ml.Recombinant mouse GM-CSF was added to the cells at a final concentrationof 200 U/ml on days 1, 2, 3, 6 and 8. On day 10, LPS at 1 μg/ml or TNF-αat 500 U/ml was added to the cells The cells were harvested on day 11.

[0190] In the absence of TNFL1, the cultured DCs triggered a 100-foldincrease in thymidine incorporation in the responding T cells. When theassay was performed in the presence of TNFL1, the increase in thymidineincorporation was only 10-fold, showing that TNFL1 is able to reduce theT cell stimulation induced by allogeneic antigen presenting cells.

[0191] Overall, these results indicate that TNFL1 is able to induce adecrease in thymidine incorporation in activated B and T cells. Thiseffect could be due to a direct inhibition of proliferation or to theinduction of apoptosis of B and T cells.

[0192] In order to elucidate the exact mechanism of action of TNFL1 onactivated T cells, a TUNEL assay (TdT-mediated dUTP-FITC nick-endlabeling; Boehringer Mannheim) was performed according to themanufacturer's specifications. Briefly, treated cells were washed twicein PBS supplemented with 1% bovine serum albumin, fixed with 0.4%paraformaldehyde, and permeabilized in 0.1% Triton X-100 in 0.1% sodiumcitrate. TUNEL staining was performed with 50 μl of TUNEL mix per sampleat 37° C. for 1 hour. Negative controls were treated similarly but werenot exposed to the enzyme.

[0193] After incubation of the T cells with TNFL1, the amount ofapoptotic cells measured by flow cytometry analysis (FIG. 5D). Theblasted cells, visualized by forward size scattering (region R1), almosttotally disappeared after addition of TNFL1. At the same time, theamount of apoptotic, FITC-positive cells (region R2) dramaticallyincreased. No apoptotic cells were detected in the absence of dUTP-FITC,whether or not TNFL1 was added.

[0194] These results strongly suggest that TNFL1 is able to induceapoptotic cell death of blasting as well as of slightly activated Tcells.

EXAMPLE 7

[0195] This example demonstrates that TNFL1 activates NFκB and leads toits translocation to the nucleus.

[0196] TNF activates the transcription factor NFκB. NFκB represents afamily of related proteins involved in the transcriptional control ofnumerous cellular genes such as interleukin-2, interleukin-2 receptor,β-interferon, granulocyte macrophage colony-stimulating factor,histocompatibility antigens, TNF, and lymphotoxin a. We thereforedecided to test whether TNFL1 could induce a similar intracellularresponse in Jurkat cells.

[0197] Jurkat cells (10⁷) were incubated for one hour with PMA at aconcentration of 1 μg/ml or TNFL1 at a concentration of 3 μg/ml in 1 mlof RPMI and 10% FBS. Nuclear extracts were prepared by centrifuging thecells at 2000 rpm and resuspending the pellet in 1 ml of cold buffer A(10 mM Hepes, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF). After asecond centrifugation at 2000 rpm, the pellet was resuspended in 20 μlof buffer A and 0.1% NP40, incubated for 10 minutes on ice, and thencentrifuged. The pellet was resuspended in 15 μl of buffer C (20 mMHepes, pH 7.9, 0.42 M NaCl, 1.5 MM MgCl₂, 0.2 mM EDTA, pH 7.4, 25%glycerol, 0.5 mM PMSF, 0.5 mM DTT, 50 μg/ml leupeptin, 50 μg/mlpepstatin, and 78 μg/ml benzamidin). The mixture was incubated for 15minutes on ice, then centrifuged for 10 minutes at 14,000 rpm at 4° C.The supernatant was diluted in 75 μl of buffer D (20 mM Hepes, pH 7.9,20% glycerol, 0.2 mM EDTA, pH 7.4, 50 mM KCl, 0.5 mM DTT, and 0.5 mMPMSF).

[0198] Five μg of nuclear extract was incubated for 10 minutes at 4° C.in the presence of 5 μg of polydIdc and wild-type or mutated competitoroligonucleotide (2 ng to 200 ng). Two-tenths of a nanogram ofpolynucleotide kinase-radiolabeled probe was added to the reaction, andthe mixture was incubated at room temperature for 30 min.

[0199] The mixture was loaded on a 5% acrylamide 60:1×linked gel inTris-glycine buffer, pH 8.3, and analyzed in an electrophoretic mobilityshift assay. Specific bands, identified by competition with wild-type ormutated NFκB binding sites, were observed with both PMA- and TNFL1-treated extracts (FIG. 5E), indicating that TNFL1 is indeed able toactivate NFκB and leads to its translocation to the nucleus.

[0200] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

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Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 SerLeu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280285 2 153 PRT human 2 Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu Cys GlyGlu Arg Glu Glu 1 5 10 15 Glu Ala Arg Ala Cys His Ala Thr His Asn ArgAla Cys Arg Cys Arg 20 25 30 Thr Gly Phe Phe Ala His Ala Gly Phe Cys LeuGlu His Ala Ser Cys 35 40 45 Pro Pro Gly Ala Gly Val Ile Ala Pro Gly ThrPro Ser Gln Asn Thr 50 55 60 Gln Cys Gln Pro Cys Pro Pro Gly Thr Phe SerAla Ser Ser Ser Ser 65 70 75 80 Ser Glu Gln Cys Gln Pro His Arg Asn CysThr Ala Leu Gly Leu Ala 85 90 95 Leu Asn Val Pro Gly Ser Ser Ser His AspThr Leu Cys Thr Ser Cys 100 105 110 Thr Gly Phe Pro Leu Ser Thr Arg ValPro Gly Ala Glu Glu Cys Glu 115 120 125 Arg Ala Val Ile Asp Phe Val AlaPhe Gln Asp Ile Ser Ile Lys Arg 130 135 140 Leu Gln Arg Leu Leu Gln AlaLeu Glu 145 150 3 210 PRT human 3 Met Ala Leu Lys Val Leu Pro Leu HisArg Thr Val Leu Phe Ala Ala 1 5 10 15 Ile Leu Phe Leu Leu His Leu AlaCys Lys Val Ser Cys Glu Thr Gly 20 25 30 Asp Cys Arg Gln Gln Glu Phe LysAsp Arg Ser Gly Asn Cys Val Leu 35 40 45 Cys Lys Gln Cys Gly Pro Gly MetGlu Leu Ser Lys Glu Cys Gly Phe 50 55 60 Gly Tyr Gly Glu Asp Ala Gln CysVal Pro Cys Arg Pro His Arg Phe 65 70 75 80 Lys Glu Asp Trp Gly Phe GlnLys Cys Lys Pro Cys Ala Asp Cys Ala 85 90 95 Leu Val Asn Arg Phe Gln ArgAla Asn Cys Ser His Thr Ser Asp Ala 100 105 110 Val Cys Gly Asp Cys LeuPro Gly Phe Tyr Arg Lys Thr Lys Leu Val 115 120 125 Gly Phe Gln Asp MetGlu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro 130 135 140 Tyr Glu Pro HisCys Thr Ser Lys Val Asn Leu Val Lys Ile Ser Ser 145 150 155 160 Thr ValSer Ser Pro Arg Asp Thr Ala Val Ala Ala Val Ile Cys Ser 165 170 175 AlaLeu Ala Thr Val Leu Leu Ala Cys Ser Ser Cys Val Ser Ser Thr 180 185 190Ala Arg Gly Ser Ser Trp Arg Arg Asn Pro Ala Val Ser Ser His Pro 195 200205 Ser Val 210 4 151 PRT human 4 Met Ala Leu Lys Val Leu Pro Leu HisArg Thr Val Leu Phe Ala Ala 1 5 10 15 Ile Leu Phe Leu Leu His Leu AlaCys Lys Val Ser Cys Glu Thr Gly 20 25 30 Asp Cys Ser Arg Gln Gln Glu PheLys Asp Arg Ser Gly Asn Cys Val 35 40 45 Leu Cys Lys Gln Cys Gly Pro GlyMet Glu Leu Ser Lys Glu Cys Gly 50 55 60 Phe Gly Tyr Gly Glu Asp Ala GlnCys Val Pro Cys Arg Pro His Arg 65 70 75 80 Phe Lys Glu Asp Trp Gly PheGln Lys Cys Lys Pro Cys Ala Asp Cys 85 90 95 Ala Leu Val Asn Arg Phe GlnArg Ala Asn Cys Ser His Thr Ser Asp 100 105 110 Ala Val Cys Gly Asp CysLeu Pro Gly Phe Tyr Arg Lys Thr Lys Leu 115 120 125 Val Gly Phe Gln AspMet Glu Cys Val Pro Cys Gly Asp Pro Pro Pro 130 135 140 Pro Tyr Glu ProHis Cys Glu 145 150 5 205 PRT human 5 Met Val Gln Leu Thr Gln Gln ThrGlu Leu Gln Ser Leu Arg Arg Glu 1 5 10 15 Val Ser Arg Leu Gln Gly ThrGly Gly Pro Ser Gln Asn Gly Glu Gly 20 25 30 Tyr Pro Trp Gln Ser Leu ProGlu Gln Ser Ser Asp Ala Leu Glu Ala 35 40 45 Trp Glu Asn Gly Glu Arg SerArg Lys Arg Arg Ala Val Leu Thr Gln 50 55 60 Lys Gln Lys Lys Gln His SerVal Leu His Leu Val Pro Ile Asn Ala 65 70 75 80 Thr Ser Lys Asp Asp SerAsp Val Thr Glu Val Met Trp Gln Pro Ala 85 90 95 Leu Arg Arg Gly Arg GlyLeu Gln Ala Gln Gly Tyr Gly Val Arg Ile 100 105 110 Gln Asp Ala Gly ValTyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp 115 120 125 Val Thr Phe ThrMet Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg 130 135 140 Gln Glu ThrLeu Phe Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp 145 150 155 160 ArgAla Tyr Asn Ser Gln Tyr Ser Ala Gly Val Pro His Leu His Gln 165 170 175Gly Asp Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn 180 185190 Leu Ser Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu 195 200 205 61284 DNA human 6 accggtccgg aattcccggg tcgacccacg cgtccgccca cgcgtccgagaagactttga 60 aattcttaca aaaactgaaa gtgaaatgag gaagacagat tgagcaatccaatcggaggg 120 taaatgccag caaacctact gtacagtagg ggtagagatg cagaaaggcagaaaggagaa 180 aattcaggat aactctcctg aggggtgagc caagccctgc catgtagtgcacgcaggaca 240 tcaacaaaca cagataacag gaaatgatcc attccctgtg gtcacttattctaaaggccc 300 caaccttcaa agttcaagta gtgatatgga tgactccaca gaaagggagcagtcacgcct 360 tacttcttgc cttaagaaaa gagaagaaat gaaactgaag gagtgtgtttccatcctccc 420 acggaaggaa agcccctctg tccgatcctc caaagacgga aagctgctggctgcaacctt 480 gctgctggca ctgctgtctt gctgcctcac ggtggtgtct ttctaccaggtggccgccct 540 gcaaggggac ctggccagcc tccgggcaga gctgcagggc caccacgcggagaagctgcc 600 agcaggagca ggagccccca aggccggcct ggaggaagct ccagctgtcaccgcgggact 660 gaaaatcttt gaaccaccag ctccaggaga aggcaactcc agtcagaacagcagaaataa 720 gcgtgccgtt cagggtccag aagaaacagt cactcaagac tgcttgcaactgattgcaga 780 cagtgaaaca ccaactatac aaaaaggatc ttacacattt gttccatggcttctcagctt 840 taaaagggga agtgccctag aagaaaaaga gaataaaata ttggtcaaagaaactggtta 900 cttttttata tatggtcagg ttttatatac tgataagacc tacgccatgggacatctaat 960 tcagaggaag aaggtccatg tctttgggga tgaattgagt ctggtgactttgtttcgatg 1020 tattcaaaat atgcctgaaa cactacccaa taattcctgc tattcagctggcattgcaaa 1080 actggaagaa ggagatgaac tccaacttgc aataccaaga gaaaatgcacaaatatcact 1140 ggatggagat gtcacatttt ttggtgcatt gaaactgctg tgacctacttacaccatgtc 1200 tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaactgaaaatac 1260 aaaaaaaaaa aaaaaaaaaa aaaa 1284 7 459 DNA human 7ctggagcgct gccgctactg caacgtcctc tgcggggagc gtgaggagga ggcacgggct 60tgccacgcca cccacaaccg tgcctgccgc tgccgcaccg gcttcttcgc gcacgctggt 120ttctgcttgg agcacgcatc gtgtccacct ggtgccggcg tgattgcccc gggcaccccc 180agccagaaca cgcagtgcca gccgtgcccc ccaggcacct tctcagccag cagctccagc 240tcagagcagt gccagcccca ccgcaactgc acggccctgg gcctggccct caatgtgcca 300ggctcttcct cccatgacac cctgtgcacc agctgcactg gcttccccct cagcaccagg 360gtaccaggag ctgaggagtg tgagcgtgcc gtcatcgact ttgtggcttt ccaggacatc 420tccatcaaga ggctgcagcg gctgctgcag gccctcgag 459 8 893 DNA human 8tccggcgccg cggggcagga caaggggaag gaataaacac gtttggtgag agccatggca 60ctcaaggtcc tacctctaca caggacggtg ctcttcgctg ccattctctt cctactccac 120ctggcatgta aagtgagttg cgaaaccgga gattgcaggc agcaggaatt caaggatcga 180tctggaaact gtgtcctctg caaacagtgc ggacctggca tggagttgtc caaggaatgt 240ggcttcggct atggggagga tgcacagtgt gtgccctgca ggccgcaccg gttcaaggaa 300gactggggtt tccagaagtg taagccatgt gcggactgtg cgctggtgaa ccgctttcag 360agggccaact gctcacacac cagtgatgct gtctgcgggg actgcctgcc aggattttac 420cggaagacca aactggttgg ttttcaagac atggagtgtg tgccctgcgg agacccacct 480cctccctacg aaccacactg taccagcaag gtgaaccttg tgaagatctc ctccaccgtc 540tccagccctc gggacacggc ggtggctgcc gtcatctgca gtgctctggc cacggtgctg 600ctcgcctgct catcctgtgt gtcatctact gcaagaggca gttcatggag aagaaaccca 660gctgtaagct cccatccctc tgtctcactg tgaagtgagc ttgttagcat tgtcacccaa 720gagttctcaa gacacctggc tgagacctaa gacctttaga gcatcaacag ctacttagaa 780tacaagatgc aggaaaacga gcctcttcag gaatctcagg gcctcctagg gatgctggca 840aggctgtgat gtctcaaggc taccaggaaa aaataaaagt tgtctatacc cta 893 9 623 DNAhuman 9 gaggcaagat tcggcacgag ggcgtttggc gcggaagtgc taccaagctgcggaaagcgt 60 gagtctggag cacagcactg gcgagtagca ggaataaaca cgtttggtgagagccatggc 120 actcaaggtc ctacctctac acaggacggt gctcttcgct gccattctcttcctactcca 180 cctggcatgt aaagtgagtt gcgaaaccgg agattgcagg cagcaggaattcaaggatcg 240 atctggaaac tgtgtcctct gcaaacagtg cggacctggc atggagttgtccaaggaatg 300 tggcttcggc tatggggagg atgcacagtg tgtgccctgc aggccgcaccggttcaagga 360 agactggggt ttccagaagt gtaagccatg tgcggactgt gcgctggtgaaccgctttca 420 gagggccaac tgctcacaca ccagtgatgc tgtctgcggg gactgcctgccaggatttta 480 ccggaagacc aaactggttg gttttcaaga catggagtgt gtgccctgcggagacccacc 540 tcctccctac gaaccacact gtgagtgatg tgccaagtgg cagcagacctttaaaaaaaa 600 aagaaaaaaa aacaaacaaa aac 623 10 1260 DNA human 10cttcctagag ggactggaac ctaattctcc tgaggctgag ggagggtgga gggtctcaag 60gcaacgctgg ccccacgacg gagtgccagg agcactaaca gtacccttag cttgctttcc 120tcctccctcc tttttatttt caagttcctt tttatttctc cttgcgtaac aaccttcttc 180ccttctgcac cactgcccgt acccttaccc gccccgccac ctccttgcta ccccactctt 240gaaaccacag ctgttggcag ggtccccagc tcatgccagc ctcatctcct ttcttgctag 300cccccaaagg cctccaggca acatgggggg cccagtcaga gagccggcac tctcagttgc 360cctctggttg agttgggggg cagctctggg ggccgtggtt tgtgcatggt tcagctgacc 420caacaaacag agctgcagag cctcaggaga gaggtgagcc ggctgcaggg gacaggaggc 480ccctcccaga atggggaagg gtatccctgg cagagtctcc cggagcagag ttccgatgcc 540ctggaagcct gggagaatgg ggagagatcc cggaaaagga gagcagtgct cacccaaaaa 600cagaagaagc agcactctgt cctgcacctg gttcccatta acgccacctc caaggatgac 660tccgatgtga cagaggtgat gtggcaacca gctcttaggc gtgggagagg cctacaggcc 720caaggatatg gtgtccgaat ccaggatgct ggagtttatc tgctgtatag ccaggtcctg 780tttcaagacg tgactttcac catgggtcag gtggtgtctc gagaaggcca aggaaggcag 840gagactctat tccgatgtat aagaagtatg ccctcccacc cggaccgggc ctacaacagc 900tgctatagcg caggtgtctt ccatttacac caaggggata ttctgagtgt cataattccc 960cgggcaaggg cgaaacttaa cctctctcca catggaacct tcctggggtt tgtgaaactg 1020tgattgtgtt ataaaaagtg gctcccagct tggaagacca gggtgggtac atactggaga 1080cagccaagag ctgagtatat aaaggagagg gaatgtgcag gaacagaggc atcttcctgg 1140gtttggctcc ccgttcctca cttttccctt ttcattccca ccccctagac tttgatttta 1200cggatatctt gcttctgttc cccatggagc tccgaattct tgcgtgtgtg tagatgaggg 126011 17 DNA human 11 aggtccatgt ctttggg 17 12 18 DNA human 12 ggggatgaattgagtctg 18 13 18 DNA human 13 gtgccctcga agaaaaag 18 14 17 DNA human 14gcaagttgga gttcatc 17 15 6 PRT human 15 Glu Tyr Met Pro Thr Asp 1 5 16 6PRT human 16 Glu Tyr Met Pro Thr Glu 1 5 17 299 PRT Homo sapien 17 MetArg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu Cys Leu Val Leu 1 5 10 15Ala Leu Pro Ala Leu Leu Pro Val Pro Ala Val Arg Gly Val Ala Glu 20 25 30Thr Pro Thr Tyr Pro Trp Arg Asp Ala Glu Thr Gly Glu Arg Leu Val 35 40 45Cys Ala Gln Cys Pro Pro Gly Thr Phe Val Gln Arg Pro Cys Arg Arg 50 55 60Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln 65 70 7580 Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu Cys Gly 85 9095 Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala Thr His Asn Arg Ala 100105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His Ala Gly Phe Cys Leu Glu115 120 125 His Ala Ser Cys Pro Pro Gly Ala Gly Val Ile Ala Pro Gly ThrPro 130 135 140 Ser Gln Asn Thr Gln Cys Gln Pro Cys Pro Pro Gly Thr PheSer Ala 145 150 155 160 Ser Ser Ser Ser Glu Gln Cys Gln Pro His Arg AsnCys Thr Ala Leu 165 170 175 Gly Leu Ala Leu Asn Val Pro Gly Ser Ser SerHis Asp Thr Leu Cys 180 185 190 Thr Ser Cys Thr Gly Phe Pro Leu Ser ThrArg Val Pro Gly Ala Glu 195 200 205 Glu Cys Glu Arg Ala Val Ile Asp PheVal Ala Phe Gln Asp Ile Ser 210 215 220 Ile Lys Arg Leu Gln Arg Leu LeuGln Ala Leu Glu Ala Pro Glu Gly 225 230 235 240 Trp Gly Pro Thr Pro ArgAla Gly Arg Ala Ala Leu Gln Leu Lys Leu 245 250 255 Arg Arg Arg Leu ThrGlu Leu Leu Gly Ala Gln Asp Gly Ala Leu Leu 260 265 270 Val Arg Leu LeuGln Ala Leu Arg Val Ala Arg Met Pro Gly Leu Glu 275 280 285 Arg Ser ValArg Glu Arg Phe Leu Pro Val His 290 295 18 1347 DNA Homo sapien 18ccgacacacc aggctgcctg ggctggtccc tggctggtga ggcccctccc agaaccaccc 60ttggactgag ctctggggag ggatggtacc aggtgggtga ggggggctgc ctggggaggg 120aggggttcct atggggcgtg gcgaggctgg cccagccctc tccccgccca tatatgtagg 180gcagcagcag gatgggcttc tggacttggg cggcccctcc gcaggcggac cgggggcaaa 240ggaggtggca tgtcggtcag gcacagcagg gtcctgtgtc cgcgctgagc cgcgctctcc 300ctgctccagc aaggaccatg agggcgctgg aggggccagg cctgtcgctg ctgtgcctgg 360tgttggcgct gcctgccctg ctgccggtgc cggctgtacg cggagtggca gaaacaccca 420cctacccctg gcgggacgca gagacagggg agcggctggt gtgcgcccag tgccccccag 480gcacctttgt gcagcggccg tgccgccgag acagccccac gacgtgtggc ccgtgtccac 540cgcgccacta cacgcagttc tggaactacc tggagcgctg ccgctactgc aacgtcctct 600gcggggagcg tgaggaggag gcacgggctt gccacgccac ccacaaccgt gcctgccgct 660gccgcaccgg cttcttcgcg cacgctggtt tctgcttgga gcacgcatcg tgtccacctg 720gtgccggcgt gattgccccg ggcaccccca gccagaacac gcagtgccag ccgtgccccc 780caggcacctt ctcagccagc agctccagct cagagcagtg ccagccccac cgcaactgca 840cggccctggg cctggccctc aatgtgccag gctcttcctc ccatgacacc ctgtgcacca 900gctgcactgg cttccccctc agcaccaggg taccaggagc tgaggagtgt gagcgtgccg 960tcatcgactt tgtggctttc caggacatct ccatcaagag gctgcagcgg ctgctgcagg 1020ccctcgaggc cccggagggc tggggtccga caccaagggc gggccgcgcg gccttgcagc 1080tgaagctgcg tcggcggctc acggagctcc tgggggcgca ggacggggcg ctgctggtgc 1140ggctgctgca ggcgctgcgc gtggccagga tgcccgggct ggagcggagc gtccgtgagc 1200gcttcctccc tgtgcactga tcctggcccc ctcttattta ttctacatcc ttggcacccc 1260acttgcactg aaagaggctt ttttttaaat agaagaaatg aggtttctta aagcttaaaa 1320aaaaaaaaaa aaaaaaaaaa aaaaaaa 1347 19 1859 DNA Homo sapien 19 ggaggtggcatgtcggtcag gcacagcagg gtcctgtgtc cgcgctgagc cgcgctctcc 60 ctgctccagcaaggaccatg agggcgctgg aggggccagg cctgtcgctg ctgtgcctgg 120 tgttggcgctgcctgccctg ctgccggtgc cggctgtacg cggagtggca gaaacaccca 180 cctacccctggcgggacgca gagacagggg agcggctggt gtgcgcccag tgccccccag 240 gcacctttgtgcagcggccg tgccgccgag acagccccac gacgtgtggc ccgtgtccac 300 cgcgccactacacgcagttc tggaactacc tggagcgctg ccgctactgc aacgtcctct 360 gcggggagcgtgaggaggag gcacgggctt gccacgccac ccacaaccgt gcctgccgct 420 gccgcaccggcttcttcgcg cacgctggtt tctgcttgga gcacgcatcg tgtccacctg 480 gtgccggcgtgattgccccg ggcaccccca gccagaacac gcagtgccag ccgtgccccc 540 caggcaccttctcagccagc agctccagct cagagcagtg ccagccccac cgcaactgca 600 cggccctgggcctggccctc aatgtgccag gctcttcctc ccatgacacc ctgtgcacca 660 gctgcactggcttccccctc agcaccaggg taccaggtga gccagaggcc tgagggggca 720 gcacactgcaggccaggccc acttgtgccc tcactcctgc ccctgcacgt gcatctagcc 780 tgaggcatgccagctggctc tgggaagggg ccacagtgga tttgaggggt caggggtccc 840 tccactagatccccaccaag tctgccctct caggggtggc tgagaatttg gatctgagcc 900 agggcacagcctcccctgga gagctctggg aaagtgggca gcaatctcct aactgcccga 960 ggggaaggtggctggctcct ctgacacggg gaaaccgagg cctgatggta attctcctaa 1020 ctgcctgagaggaaggtggc tgcctcctct gacatgggga aaccgaggcc caatgttaac 1080 cactgttgagaagtcacagg gggaagtgac ccccttaaca tcaagtcagg tccggtccat 1140 ctgcaggtcccaactcgccc cttccgatgg cccaggagcc ccaagccctt gcctgggccc 1200 ccttgcctcttgcagccaag gtccgagtgg ccgctcctgc cccctaggcc tttgctccag 1260 ctctctgaccgaaggctcct gccccttctc cagtccccat cgttgcactg ccctctccag 1320 cacggctcactgcacaggga tttctctctc ctgcaaaccc cccgagtggg gcccagaaag 1380 cagggtacctggcagccccc gccagtgtgt gtgggtgaaa tgatcggacc gctgcctccc 1440 caccccactgcaggagctga ggagtgtgag cgtgccgtca tcgactttgt ggctttccag 1500 gacatctccatcaagaggct gcagcggctg ctgcaggccc tcgaggcccc ggagggctgg 1560 ggtccgacaccaagggcggg ccgcgcggcc ttgcagctga agctgcgtcg gcggctcacg 1620 gagctcctgggggcgcagga cggggcgctg ctggtgcggc tgctgcaggc gctgcgcgtg 1680 gccaggatgcccgggctgga gcggagcgtc cgtgagcgct tcctccctgt gcactgatcc 1740 tggccccctcttatttattc tacatccttg gcaccccact tgcactgaaa gaggcttttt 1800 tttaaatagaagaaatgagg tttcttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1859 20 211 PRTHomo sapien 20 Met Arg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu Cys LeuVal Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro Val Pro Ala Val Arg GlyVal Ala Glu 20 25 30 Thr Pro Thr Tyr Pro Trp Arg Asp Ala Glu Thr Gly GluArg Leu Val 35 40 45 Cys Ala Gln Cys Pro Pro Gly Thr Phe Val Gln Arg ProCys Arg Arg 50 55 60 Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg HisTyr Thr Gln 65 70 75 80 Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Cys AsnVal Leu Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala ThrHis Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His AlaGly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro Pro Gly Ala Gly ValIle Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr Gln Cys Gln Pro CysPro Pro Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser Ser Ser Ser Glu GlnCys Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu Gly Leu Ala Leu AsnVal Pro Gly Ser Ser Ser His Asp Thr Leu 180 185 190 Cys Thr Ser Cys ThrGly Phe Pro Leu Ser Thr Arg Val Pro Gly Glu 195 200 205 Pro Glu Ala 21021 145 PRT Homo sapien 21 Thr Val Thr Gln Asp Cys Leu Gln Leu Ile AlaAsp Ser Glu Thr Pro 1 5 10 15 Thr Ile Gln Lys Gly Ser Tyr Thr Phe ValPro Trp Leu Leu Ser Phe 20 25 30 Lys Arg Gly Ser Ala Leu Glu Glu Lys GluAsn Lys Ile Leu Val Lys 35 40 45 Glu Thr Gly Tyr Phe Phe Ile Tyr Gly GlnVal Leu Tyr Thr Asp Lys 50 55 60 Thr Tyr Ala Met Gly His Leu Ile Gln ArgLys Lys Val His Val Phe 65 70 75 80 Gly Asp Glu Leu Ser Leu Val Thr LeuPhe Arg Cys Ile Gln Asn Met 85 90 95 Pro Glu Thr Leu Pro Asn Asn Ser CysTyr Ser Ala Gly Ile Ala Lys 100 105 110 Leu Glu Glu Gly Asp Glu Leu GlnLeu Ala Ile Pro Arg Glu Asn Ala 115 120 125 Gln Ile Ser Leu Asp Gly AspVal Thr Phe Phe Gly Ala Leu Lys Leu 130 135 140 Leu 145 22 141 PRT Homosapien 22 Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn SerArg 1 5 10 15 Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val LeuLeu Ser 20 25 30 Gly Val Lys Tyr Lys Lys Gly Gly Leu Val Ile Asn Glu ThrGly Leu 35 40 45 Tyr Phe Val Tyr Ser Lys Val Tyr Phe Arg Gly Gln Ser CysAsn Asn 50 55 60 Leu Pro Leu Ser His Lys Val Tyr Met Arg Asn Ser Lys TyrPro Gln 65 70 75 80 Asp Leu Val Met Met Glu Gly Lys Met Met Ser Tyr CysThr Thr Gly 85 90 95 Gln Met Trp Ala Arg Ser Ser Tyr Leu Gly Ala Val PheAsn Leu Thr 100 105 110 Ser Ala Asp His Leu Tyr Val Asn Val Ser Glu LeuSer Leu Val Asn 115 120 125 Phe Glu Glu Ser Gln Thr Phe Phe Gly Leu TyrLys Leu 130 135 140 23 147 PRT Homo sapien 23 Ser Thr Leu Lys Pro AlaAla His Leu Ile Gly Asp Pro Ser Lys Gln 1 5 10 15 Asn Ser Leu Leu TrpArg Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp 20 25 30 Gly Phe Ser Leu SerAsn Asn Ser Leu Leu Val Pro Thr Ser Gly Ile 35 40 45 Tyr Phe Val Tyr SerGln Val Val Phe Ser Gly Lys Ala Tyr Ser Pro 50 55 60 Lys Ala Thr Ser SerPro Leu Tyr Leu Ala His Glu Val Gln Leu Phe 65 70 75 80 Ser Ser Gln TyrPro Phe His Val Pro Leu Leu Ser Ser Gln Lys Met 85 90 95 Val Tyr Pro GlyLeu Gln Glu Pro Trp Leu His Ser Met Tyr His Gly 100 105 110 Ala Ala PheGln Leu Thr Gln Gly Asp Gln Leu Ser Thr His Thr Asp 115 120 125 Gly IlePro His Leu Val Leu Ser Pro Ser Thr Val Phe Phe Gly Ala 130 135 140 PheAla Leu 145 24 161 PRT Homo sapien 24 Ser Pro Gly Leu Pro Ala Ala HisLeu Ile Gly Ala Pro Leu Lys Gly 1 5 10 15 Gln Gly Leu Gly Trp Glu ThrThr Lys Glu Gln Ala Phe Leu Thr Ser 20 25 30 Gly Thr Gln Phe Ser Asp AlaGlu Gly Leu Ala Leu Pro Gln Asp Gly 35 40 45 Leu Tyr Tyr Leu Tyr Cys LeuVal Gly Tyr Arg Gly Arg Ala Pro Pro 50 55 60 Gly Gly Gly Asp Pro Gln GlyArg Ser Val Thr Leu Arg Ser Ser Leu 65 70 75 80 Tyr Arg Ala Gly Gly AlaTyr Gly Pro Gly Thr Pro Glu Leu Leu Leu 85 90 95 Glu Gly Ala Glu Thr ValThr Pro Val Leu Asp Pro Ala Arg Arg Gln 100 105 110 Gly Tyr Gly Pro LeuTrp Tyr Thr Ser Val Gly Phe Gly Gly Leu Val 115 120 125 Gln Leu Arg ArgGly Glu Arg Val Tyr Val Asn Ile Ser His Pro Asp 130 135 140 Met Val AspPhe Ala Arg Gly Lys Thr Phe Phe Gly Ala Val Met Val 145 150 155 160 Gly25 150 PRT Homo sapien 25 Pro Ser Asp Lys Pro Val Ala His Val Val AlaAsn Pro Gln Ala Glu 1 5 10 15 Gly Gln Leu Gln Trp Leu Asn Arg Arg AlaAsn Ala Leu Leu Ala Asn 20 25 30 Gly Val Glu Leu Arg Asp Asn Gln Leu ValVal Pro Ser Glu Gly Leu 35 40 45 Tyr Leu Ile Tyr Ser Gln Val Leu Phe LysGly Gln Gly Cys Pro Ser 50 55 60 Thr His Val Leu Leu Thr His Thr Ile SerArg Ile Ala Val Ser Tyr 65 70 75 80 Gln Thr Lys Val Asn Leu Leu Ser AlaIle Lys Ser Pro Cys Gln Arg 85 90 95 Glu Thr Pro Glu Gly Ala Glu Ala LysPro Trp Tyr Glu Pro Ile Tyr 100 105 110 Leu Gly Gly Val Phe Gln Leu GluLys Gly Asp Arg Leu Ser Ala Glu 115 120 125 Ile Asn Arg Pro Asp Tyr LeuAsp Phe Ala Glu Ser Gly Gln Val Tyr 130 135 140 Phe Gly Ile Ile Ala Leu145 150

1. An isolated human protein having an amino acid sequence which is atleast 85% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 2, 17, and 20, wherein percent identity isdetermined using a Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of
 1. 2. The isolated human protein of claim 1 which has anamino acid sequence selected from the group consisting of SEQ ID NOS:1,2, 17, and
 20. 3. A fusion protein comprising a first protein segmentand a second protein segment fused together by means of a peptide bond,wherein the first protein segment consists of a protein having an aminoacid sequence selected from the group consisting of SEQ ID NOS:1, 2, and17.
 4. A preparation of antibodies which specifically bind to a proteinhaving an amino acid sequence selected from the group consisting of SEQID NOS:1, 2, 17, and
 20. 5. A cDNA molecule which encodes a proteinhaving an amino acid sequence which is at least 85% identical to anamino acid sequence selected from the group consisting of SEQ ID NOS:1,2, 17, and 20, wherein percent identity is determined using aSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of
 1. 6. The cDNAmolecule of claim 5 which encodes an amino acid sequence selected fromthe group consisting of SEQ ID NOS:1, 2, 17, and
 20. 7. The cDNAmolecule of claim 6 which comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NOS:6, 7, 18 and
 19. 8. A cDNA moleculewhich is at least 85% identical to a nucleotide sequence selected fromthe group consisting of SEQ ID NOS:6, 7, 18, and 19, wherein percentidentity is determined using a Smith-Waterman homology search algorithmusing an affine gap search with a gap open penalty of 12 and a gapextension penalty of
 1. 9. An isolated and purified subgenomicpolynucleotide comprising a nucleotide sequence which hybridizes to anucleotide sequence selected from the group consisting of SEQ ID NOS:6,7, 18, and 19 after washing with 0.2×SSC at 65° C., wherein thenucleotide sequence encodes a protein having an amino acid sequenceselected from the group consisting of SEQ ID NOS:1, 2, and
 17. 10. Aconstruct comprising. a promoter; and a polynucleotide segment encodingan amino acid sequence selected from the group consisting of SEQ IDNOS:1, 2, 17, and 20, wherein the polynucleotide segment is locateddownstream from the promoter, wherein transcription of thepolynucleotide segment initiates at the promoter.
 11. A host cellcomprising a construct which comprises: a promoter; and a polynucleotidesegment encoding an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 2, 17, and
 20. 12. A recombinant host cellcomprising a new transcription initiation unit, wherein the newtranscription initiation unit comprises in 5′ to 3′ order: (a) anexogenous regulatory sequence, (b) an exogenous exon; and (c) a splicedonor site, wherein the new transcription initiation unit is locatedupstream of a coding sequence of a gene, wherein the coding sequence isselected from the group consisting of SEQ ID NOS:6, 7, 18, and 19,wherein the exogenous regulatory sequence controls transcription of thecoding sequence of the gene.
 13. A method of screening for a compoundcapable of modulating cell death inducing activity of a protein,comprising the steps of: incubating a first population of cells and aprotein in the presence of a test compound, wherein the proteincomprises an amino acid sequence selected from the group of amino acidsequences shown in SEQ ID NOS:1-5, 17, and 20; incubating a secondpopulation of cells and the protein in the absence of a test compound;and determining viability of the first and second populations, wherein atest compound which increases or decreases viability of the firstpopulation relative to the second population is identified as capable ofmodulating the cell death inducing activity of the protein.
 14. Themethod of claim 13 wherein the protein is provided to the first andsecond populations of cells by transfecting the first and secondpopulations of cells with a polynucleotide encoding the protein.
 15. Themethod of claim 13 wherein the polynucleotide comprises a nucleotidesequence selected from the group consisting of SEQ ID NOS:6-10, 18, and19.
 16. A method of identifying a binding partner of a first protein,comprising the steps of: incubating a first protein with a secondprotein, wherein the first protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1-5, 17, and 20;detecting formation of a complex between the first and second proteins,wherein formation of the complex identifies the second protein as abinding partner of the first protein.